Bacterial superantigen vaccines

ABSTRACT

The present invention relates to genetically attenuated superantigen toxin vaccines altered such that superantigen attributes are absent, however the superantigen is effectively recognized and an appropriate immune response is produced. The attenuated superantigen toxins are shown to protect animals against challenge with wild type toxin. Methods of producing and using the altered superantigen toxins are described.

Staphylococcal enterotoxins (SEs) A through E are the most commom causeof food poisoning [Bergdoll, M. S.(1983) In Easom CSF, Aslam C., eds.Staphylococci and staphylococcal infections. London: Academic Press, pp559-598] and are associated with several serious diseases [Schlievert,P. M. (1993) J. Infect. Dis. 167: 997-1002; Ulrich et al. (1995) TrendsMicrobiol. 3: 463-468], such as bacterial arthritis [Schwab et al.(1993) J. Immunol. 150; 4151-4159; Goldenberg et al. (1985) N. Engl. J.Med. 312: 764-771], other autoimmune disorders [Psnett, D. N. (1993)Semin. Immunol. 5: 65-72], and toxic shock syndrome [Schlieverst, P. M.(1986) Lancet 1: 1149-1150; Bohach et al. (1990) Crit. Rev. Microbiol.17: 251-272]. The nonenterotoxic staphylococcal superantigen toxic shocksyndrome toxin-1 (TSST-1) was first identified as a causative agent ofmenstrual-associated toxic shock syndrome [Schlievert et al. (1981) J.Infect. Dis. 143: 509-516]. Superantigen-producing Staphylococcus aureusstrains are also linked to Kawasaki syndrome, an inflammatory disease ofchildren [Leung et al. (1993) Lancet 342: 1385-1388].

The staphylococcal enterotoxins A-E, toxic shock syndrome toxin-1(TSST-1), and streptococcal pyrogenic exotoxins A-C are soluble 23-29-kDproteins commonly referred to as bacterial superantigens (SAgs).Bacterial superantigens are ligands for both major histocompatibilitycomplex (MHC) class II molecules, expressed on antigen-presenting cells,and the variable portion of the T cell antigen receptor β chain (TCR Vβ)[Choi et al. (1989) Proc. Natl. Acad. Sci. USA86:8941-8945; Fraser, J.D. (1989) Nature 339:221-223; Marrack et al. (1990) Science 248:705-711; Herman et al. (1991) Annu. Rev. Immunol. 9: 745-772; Mollick etal. (1989) Science 244:817-820].

Each bacterial superantigen has a distinct affinity to a set of TCR Vβ,and coligation of the MHC class II molecule polyclonally stimulates Tcells [White et al. (1989) Cell 56: 27-35; Kappler et al. (1989) Science244: 811-813; Takimoto et al. (1990) Eur J. Immunol. 140: 617-621].Pathologically elevated levels of cytokines that are produced byactivated T cells are the probable cause of toxic shock symptoms [Calsonet al. (1985) Cell. Immunol. 96: 175-183; Stiles et al. (1993) Infect.Immun. 61: 5333-5338]. In addition, susceptibility to lethalgram-negative endotoxin shock is enhanced by several bacterialsuperantigens [Stiles, et al., supra]. Although antibodies reactive withsuperantigens are present at low levels in human sera [Takei et al.(1993) J. Clin. Invest. 91: 602-607], boosting antibody titers byspecific immunization may be efficacious for patients at risk for toxicshock syndrome and the other disorders of common etiology.

A vaccine approach to controlling bacterial superantigen-associateddiseases presents a unique set of challenges. Acute exposure tobacterial superantigens produces T cell anergy, a state of specificnon-responsiveness [Kawabe et al. (1991) Nature 349: 245-248], yet Tcell help is presumably a requirement for mounting an antibody response.

Presently, the only superantigen vaccines available are chemicallyinactivated toxoids from different bacteria which have severaldisadvantages. The chemical inactivation process can be variable foreach production lot making the product difficult to characterize. Thechemical used for inactivation, (e.g. formaldehyde), is often toxic anddoes not negate the possibility of reversion of the inactivatedsuperantigen to an active form. In addition, the yields of wild-typetoxin from bacterial strains used for making toxoids are often low.

SUMMARY OF THE INVENTION

The present invention relates to a vaccine which overcomes thedisadvantages of the chemically inactivated toxoids described above. Thesuperantigen vaccine(s) of the present invention is/are designed toprotect individuals against the pathologies resulting from exposure toone or several related staphylococcal and streptococcal toxins. Thesuperantigen vaccine is comprised of a purified protein product that isgenetically attenuated by DNA methodologies such that superantigenattributes are absent, however the superantigen is effectivelyrecognized by the immune system and an appropriate antibody response isproduced.

Specifically, the vaccine of the present invention is a product ofsite-directed mutagenesis of the DNA coding sequences of superantigentoxins resulting in a disruption of binding to both the MHC class IIreceptor and to the T-cell antigen receptor. A comprehensive study ofthe relationships of the superantigen structures of TSST-1,streptococcal pyrogenic exotoxin-A (SPEa), staphylococcal enterotoxin B(SEB), and staphylococcal enterotoxin A, to receptor binding wereundertaken to provide insight into the design of the vaccine. From thesestudies, critical amino acid residues of the toxin responsible forbinding the superantigen to the human MHC receptor were defined.Site-directed mutagenesis of the gene encoding the toxin and expressionof the new protein product resulted in a superantigen toxin withdisrupted binding to the MHC receptors.

Therefore, it is an object of the present invention to provide asuperantigen toxin DNA fragment which has been genetically altered suchthat binding of the encoded altered toxin to the MHC class II or T-cellantigen receptor is disrupted. Such a DNA fragment is useful in theproduction of a vaccine against superantigen toxin infections.

It is another object of the present invention to provide a superantigentoxin amino acid sequence which has been altered such that the bindingof the encoded altered toxin to the MHC class II or T-cell antigenreceptor is disrupted. Such a sequence is useful for the production of asuperantigen toxin vaccine.

It is another object of the invention to provide a recombinant vectorcomprising a vector and the DNA fragment described above.

It is a further object of the present invention to provide host cellstransformed with the above-described recombinant DNA constructs. Hostcells include cells of other prokaryotic species or eukaryotic plant oranimal species, including yeasts, fungi, plant culture, mammalian andnonmammalian cell lines, insect cells and transgenic plants or animals.

It is another object of the present invention to provide a method forproducing altered superantigen toxin with disrupted MHC class II andT-cell antigen receptor binding which comprises culturing a host cellunder conditions such that a recombinant vector comprising a vector andthe DNA fragment described above is expressed and altered superantigentoxin is thereby produced, and isolating superantigen toxin for use as avaccine against superantigen toxin-associated bacterial infections andas a diagnostic reagent.

It is still another object of the invention to provide a purifiedaltered superantigen toxin useful as a vaccine and as a diagnosticagent.

It is another object of the invention to provide a purified alteredsuperantigen toxin for the therapeutic stimulation of, or other in vivomanipulation of, selective T cell subsets, or ex vivo manipulation of Tcells for in vivo therapeutic purposes in mammals. Diseases, such asautoimmunity, wherein T-cell responses of limited diversity(oligoclonal) are evident. Altered superantigens may be used totherapeutically inactivate (induce anergy in) T cells in diseaseswherein oligoclonal T-cell responses are evident such as autoimmunediseases, for example. For diseases in which specific T-cell subsets arenot active or are anergetic, altered superantigens may be used totherapeutically stimulate these T cells. Such disease include, but arenot limited to, infectious diseases and cancers wherein specific subsetsof cytotoxic or helper T cells are inactivated or are otherwise unableto respond normally to the antigenic stimulation of the disease moiety.

It is a further object of the present invention to provide an antibodyto the above-described altered superantigen toxin for use as atherapeutic agent and as a diagnostic agent.

It is yet another object of the invention to provide a superantigentoxin vaccine comprising an altered superantigen toxin effective for theproduction of antigenic and immunogenic response resulting in theprotection of an animal against superantigen toxin infection.

It is a further object of the invention to provide a multivalentsuperantigen toxin vaccine comprising altered toxins from a variety ofstreptococcal and staphylococcal toxins effective for the production ofantigenic and immunogenic response resulting in the protection of ananimal against infection with bacterial superantigen toxin-expressingstrains and against other direct or indirect exposures to bacterialsuperantigen toxins such as might occur by ingestion, inhalation,injection, transdermal or other means.

It is yet another object of the present invention to provide a methodfor the diagnosis of superantigen toxin-associated bacterial infectioncomprising the steps of:

(i) contacting a sample from an individual suspected of having asuperantigen toxin-associated bacterial infection with antibodies whichrecognize superantigen toxin using antibodies generated from the alteredsuperantigen toxin; and

(ii) detecting the presence or absence of a superantigen-associatedbacterial infection by detecting the presence or absence of a complexformed between superantigen toxin in said sample and antibodies specifictherefor.

It is yet another object of the present invention to provide a methodfor the diagnosis of superantigen bacterial infection comprising thesteps of:

(i) contacting a sample from an individual suspected of having thedisease with lymphocytes which recognize superantigen toxin produced bysaid superantigen bacteria or lymphocytes which recognize alteredsuperantigen toxin; and

(ii) detecting the presence or absence of responses of lymphocytesresulting from recognition of superantigen toxin. Responses can be, forexample, measured cytokine release, increase of activation markers,mitotic activity, or cell lysis. The lymphocytes responding to thealtered superantigen toxins recognize them as recall antigens not assuperantigens, therefore the response is an indicator of prior exposureto the specific superantigen. The absence of a response may indicate noprior exposure, a defective immune response or in some cases amanifestation of T-cell anergy. Anergy is defined here asantigen-specific or a generalized non-responsiveness of subsets of Tcells.

It is a further object of the present invention to provide a diagnostickit comprising an antibody against an altered superantigen toxin andancillary reagents suitable for use in detecting the presence ofsuperantigen toxin in animal tissue or serum.

It is another object of the present invention to provide a detectionmethod for detecting superantigen toxins or antibodies to superantigentoxin in samples, said method comprising employing a biosensor approach.Such methods are known in the art and are described for example inKarlsson et al. (1991) J. Immunol. Methods 145, 229-240 and Jonsson etal. (1991) Biotechniques 11, 620-627.

It is yet another object of the present invention to provide atherapeutic method for the treatment or amelioration of symptoms ofsuperantigen-associated bacterial infection, said method comprisingproviding to an individual in need of such treatment an effective amountof sera from individuals immunized with one or more altered superantigentoxins from different bacteria in a pharmaceutically acceptableexcipient.

It is further another object of the present invention to provide atherapeutic method for the treatment or amelioration of symptoms ofsuperantigen toxin-associated bacterial infection, said methodcomprising providing to an individual in need of such treatment aneffective amount of antibodies against altered superantigen toxins in apharmaceutically acceptable excipient.

It is another object of the present invention to provide a therapeuticmethod for the treatment or amelioration of symptoms of bacterialsuperantigen toxin infection, said method comprising providing to anindividual in need of such treatment an effective amount of alteredsuperantigen from a variety of streptococcal and staphylococcal bacteriain order to inhibit adhesion of superantigen bacterial toxin to MHCclass II or T-cell receptors by competitive inhibition of theseinteractions.

It is yet another object of the present invention to provide atherapeutic method for the treatment of diseases that may not beassociated directly with superantigen toxins but which result inspecific nonresponsiveness of T-cell subsets, said method comprising theadministration of altered superantigen toxins, in vivo or ex vivo, suchthat T-cell subsets are expanded or stimulated. Diseases which causeanergy or nonresponsiveness of T-cells include, but are not limited to,infectious diseases.

It is another object of the present invention to provide a therapeuticmethod for the treatment of diseases associated with expanded orover-stimulated T-cell subsets, such as autoimmunity for example, saidmethod comprising administration of altered superantigen toxin, in vivoor ex vivo, such that anergy or inactivation of disease associatedT-cells is produced. In this case, superantigen mutants can be designedwith altered but not attenuated T-cell receptor binding, to cause anergyof only the select (i.e. 1-3) T-cell subsets that are pathologicallyactivated.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription and appended claims, and accompanying drawings where:

FIG. 1. Staphylococcal and streptococcal superantigen amino acidsequence homologies, compiled with Genetics Computers Group of Univ. ofWisconsin software.

FIG. 2. Comparison of mutant SEB and SEA biological activities.

A. SEB mutant HLA-DR1-binding; B. SEA mutant HLA-DR1-binding; C. T-cellrecognition of SEA and SEB mutants. Binding of bacterial superantigensto cell surface DR1 was measured by laser fluorescence-activated flowcytometry. A representative experiment of three performed is shown. Themutants SEA D197N, the homologous SEB D199N, and SEA L11Y had no effecton binding or T-cell recognition, and were used for controls. HumanT-cell proliferation, assessed by [³H]thymidine incorporation, wasmeasured in response to SEA (Y64A) or SEB (Y61A) mutants and controlsthat retained DR1-binding affinities. Each data point represents themean of triplicate determinations; SEM <5%.

FIG. 3. Sequence and secondary structural alignment of bacterialsuperantigen toxins. Analyses were performed with the applicationsPILEUP and PROFILE from the Computer Genetics Group (Madison, Wis.)using sequence data obtained from a variety of sources. Amino acidresidue numbering is based on the SEA sequence SEA (SEQ ID NO:17), SED(SEQ ID NO:18), SEE (SEQ ID NO:19), SEB (SEQ ID NO:20), SEC1 (SEQ IDNO:21), SEC2 (SEQ ID NO:22), SEC3 (SEQ ID NO:23), SPEa (SEQ ID NO:24)and TSST1 (SEQ ID NO:25).

FIG. 4. Detection of TNF-α (a), IL-1α (B), IL-6 (C) and IFN-γ (D) in theserum of mice injected with SEA (open circles), LPS (open triangles), orSEA plus LPS (open squares). Values for TNF-α and IL-1α represent themean of duplicate samples, with an SEM of ±5%. INF-γ and IL-6 valuesrepresent the mean of duplicate and triplicate samples, respectively.The SEMs for IFN-γ and IL-6 readings were ±5% and ±10%, respectively.

FIG. 5. Mutant SEA vaccines that have attenuated majorhistocompatibility complex class II or T-cell antigen receptor bindingdo not induce T-cell anergy. Mice were given three doses of wild type(WT) SEA or site-specific mutant vaccine, plus adjuvant. Control animalsreceived adjuvant alone or were untreated; 2 weeks after finalinjection, pooled mononuclear cells were collected from spleens of 4mice from each group. Results are represented as mean cpm (±SD) ofquadruplicate wells incubated with 100 ng/ml WT SEA for 72 h and thenpulse-labeled for 12 h with [³H]thymidine. P<0.0001 (analysis ofvariance for repeated measures comparing untreated, adjuvant, Y64A, andY92A to WT SEA group).

FIG. 6. No superantigen-induced T-cell anergy is exhibited by rhesusmonkeys immunized with the vaccine B899445. Peripheral blood lymphocyteswere incubated with titrated concentrations of wild-type superantigensfrom individual rhesus monkeys (K422 and N103) that were immunized withB899445. T-cell proliferation was assessed by [³H] thymidineincorporation. Each data point represents the mean of triplicatedeterminations; SEM <5%.

FIG. 7. Antibody responses of rhesus monkeys immunized with a combinedvaccine consisting of B899445 (SEB) and A489270 (SEA). The antibodylevels were measured by ELISA, using plates coated with SEA, SEB or SEC1as listed. Monkey G8 is a non-immunized control. SEM <5% for triplicatemeasurements.

DETAILED DESCRIPTION

The present invention relates in part to a vaccine against superantigentoxin-associated bacterial diseases. The superantigen vaccines used inthis study were developed by engineering changes in the receptor-bindingportions of superantigen toxins to reduce receptor-binding affinitiesand toxicity while maintaining antigenicity.

Five different superantigen vaccines are described in this application:staphylococcal enterotoxin A, staplylococcal enterotoxin B,staphylococcal enterotoxin C1, toxic-shock syndrome toxin-1, andstreptococcal pyrogenic exotoxin-A. For each of the superantigen toxinsabove, a comprehensive study of the relationships of the toxin proteinstructure to receptor binding was undertaken to provide insight into thedesign of the vaccine. The study employed site-directed mutagenesis oftoxin and receptor molecules, molecular modeling, protein structure andbinding studies. Following these studies, toxins were altered bysite-directed mutagenesis to attenuate MHC class II binding andbiological activity to an essentially non-specific level. The engineeredvaccines were evaluated at each stage of analysis to determine mouse andhuman T-cell reactivities in vitro, serological responses and toxicityin mice and monkeys.

In one embodiment, the present invention relates to an alteredsuperantigen toxin protein having an amino acid sequence which has beenaltered such that the binding of the toxin to the MHC class II receptoris disrupted.

Comparison of amino acid sequences (FIG. 1) suggested that bacterialsuperantigens fall into groups consisting of (1) SEA, SED and SEE, (2)SEB, staphylococcal enterotoxins C1-C3 (SEC1-3), the streptococcalpyrogenic exotoxins A (SPE-A) and C (SPE-C), (3) TSST-1 and (4) theexfoliative toxins (ETA, ETB) and streptococcal pyrogenic exotoxin B(SPE-B), which are the most distant from the others in sequence.Although not available to the inventor when the inventions were firstconceived and proof of principle was obtained, the x-raycrystallographic structures of several bacterial superantigens are nowknown. Diverse superantigens, such as SEB and TSST-1, appear to havelittle sequence in common, yet they exhibit homologous protein foldscomposed largely of β strands [Prasad, G. S. et al. (1993) Biochemistry32, 13761-13766; Acharya, R. K. et al. (1994) Nature 367, 94-97;Swaminathan, S. et al. (1992) Nature 359, 801-806] within two distinctdomains. Differences between the proteins are located primarily inhighly variable regions comprised of several surface loops, such as thedisulfide-bonded loop which is absent from TSST-1 and at the aminoterminus.

The X-ray crystal structures of SEB and TSST-1 complexed with HLA DR1are known [Kim, J. et al. (1994) Science 266, 1870-1874; Jardetzky, T.S. et al. (1994) Nature 368, 711-718]. The region of HLA DR1 thatcontacts SEB consists exclusively of a subunit surfaces. The mainregions of SEB involved are two conserved sites: a polar pocket derivedfrom three β strands of the β barrel domain and a highly solvent-exposedhydrophobic reverse turn. The polar binding pocket of SEB contains aglutamate and two tyrosines that accommodate Lys39 of the α subunit ofHLA DR1, while the hydrophobic region consists of a leucine and flankingresidues that make several contacts with the HLA DRα chain. The HLA DR1binding sites of both TSST-1 and SEB overlap significantly. Thehydrophobic binding contacts of other SAg with the HLA DRα chain havebeen proposed [Ulrich, et al. (1995). Nature, Struct. Biol 2, 554-560]to be similar to those found in SEB and TSST-1. A motif consisting of aleucine in a reverse turn [Ulrich et al. (1995), supra] is conservedamong bacterial superantigens and may provide the key determinant(hydrophobic or otherwise) for binding HLA-DR. However, TSST-1 does nothave a highly charged residue in the polar pocket that interacts withLys39 of the HLA DRα chain and uses an alternative conformationalbinding mode that allows TSST-1 to interact with HLA DR1 β-chainresidues and the carboxy-terminal region of the antigenic peptide.

Both SEA and SEE bind to the β subunit of DR by means of a single zincatom [Fraser, J. D. et al. (1992) Proc. Natl. Acad. Sci. USA 89,5507-5511]. The amino-terminal domain of SEA interfaces with the HLA DRαchain [Ulrich, et al. (1995)], while SEA C-terminal domain residuesHis187, His225 and Asp227 form a zinc-coordination complex, likely withHis-81 from the β chain of an adjoining HLA DR molecule. However, ourresults have shown that binding of superantigen to the HLA DRβ subunitdoes not directly stimulate T cells [Ulrich et al. (1995) Nature,Struct. Biol. 2, 554-560], but increases the potential of the bound SEAto interact with the α chain of another HLA DR, thus increasing thebiological potency.

A least-squares superimposition of the unbound molecules of modeled SEAand the crystal structure of SEB, aligned according to theirstructurally conserved α-helical and β-strand regions, exhibited aglobal folding pattern which is very similar. Differences between thetwo structures are calculated to be located primarily in loops of lowsequence homologies, with the largest positional deviations occurringbetween structurally conserved regions of residues 18-20, 30-32,173-181, 191-194, and the cysteine-loop region (90-111). Only one ofthese regions in SEB makes significant contact (residue Y94 [Y=tyrosine]in particular) with the HLA-DR1 molecule [Jardetzky, T. S. et al. (1994)Nature 368, 711-718].

The binding interface between SEB and HLA-DR1 consists principally oftwo structurally conserved surfaces located in the N-terminal domain: apolar binding pocket derived from three β-strand elements of theβ-barrel domain and a hydrophobic reverse turn. The binding pocket ofSEB contains residues E67 (E=Glutamic acid), Y89 (Y=Tyrosine) and Y115(Y=tyrosine), and binds K39 (K=Lysine) of the DRα subunit. The aminoacid one letter code is defined as the following: A=Alanine (Ala),I=Isoleucine (Ile), L=Leucine (Leu), M=Methionine (Met), F=Phenylalanine(Phe), P=Proline (Pro), W=Tryptophan (Trp), V=Valine (Val), N=Asparagine(Asn), C=Cysteine (Cys), Q=Glutamine (Q), G=Glycine (Gly), S=Serine(Ser), T=Threonine (Thr), Y=Tyrosine (Tyr), R=Arginine (Arg),H=Histidine (His), K=Lysine (Lys), D=Aspartic acid (Asp), and E=Glutamicacid (Glu). For SEA, the binding interface with the DR molecule ismodeled to contain a similar binding pocket consisting of residues D70,Y92 and Y108. Mutation of residue Y89 in SEB or Y92 in SEA to alanine(FIG. 2) resulted in greater than 100-fold reduction in DR1 binding. Thesubstitution of alanine for Y89 in SEB and Y92 in SEA eliminates thehydrogen bond with K39 and disrupts packing interactions with adjacentprotein residues. Modeling of the SEA mutant Y92A predicts an increasein solvent-accessible surface area for Y108 by a factor of two greaterthan the wild-type structure, allowing the formation of a hydrogen bondto the carboxylate group of D70 and thus disrupting key anchoring andrecognition points for HLA-DR1. This effect is expected to be somewhatless in SEB due to the longer side chain at E67. Substitution of SEBY115 with alanine also resulted in greater than 100-fold reduction ofbinding. In contrast, the same replacement of Y108 in SEA yielded littleto no change in DR1 binding (FIG. 2a), suggesting the primary importanceof SEA residues Y92 and D70 for stabilizing interactions with K39. TheK39 side chain of DRα forms a strong ion-pair interaction with the SEBE67 carboxylate group and hydrogen bonds with the hydroxyl groups of Y89and Y115. Substitution of SEB E67 by glutamine reduced binding affinityby greater than 100-fold (FIG. 2), reflecting the replacement of thestrong ionic bond with a weaker hydrogen bond. To optimize ion-pairinteractions of the analogous SEA site, the shorter carboxylate sidechain of D70 is predicted to shift K39 of DRα, weakening interactionswith SEA Y108. The substitution of alanine for SEA Y108 is thus moreeasily accommodated than the homologous substitution of SEB Y115,without loss in DR1 binding.

Comparisons of the polar pocket with other bacterial superantigens werethen made. SEC1-3 and SPE-A have conserved the critical DR1binding-interface residues (FIG. 1), and share with SEB and SEAsecondary structural elements of the DR1-binding surfaces. Asparagine inSED (N70) replaces the acidic side chain present in SEA, SEB, SPE-A andSEC1-3. Accordingly, for SED the salt bridge of the polar pocket islikely to be replaced by a hydrogen bond. Overall, DR1 affinities forSED and SEA appeared to be equivalent (FIG. 2b), indicating that otherinteractions may compensate for the absence in SED of the ion-pair foundin the other superantigens. For the case of TSST-1, mutating DRαresidues K39 to serine or M36 to isoleucine has been shown to greatlyreduce binding [Panina-Bordignon et al. (1992) J. Exp. Med. 176:1779-1784]. Although primarily hydrophobic, the critical TSST-1structural elements are conserved with the SEA and SEB polar bindingpocket. SEB residues Y89 and Y115 are homologous to T69 and 185 inTSST-1, respectively, and SEB E67 is replaced by I46. These TSST-1residues are positioned in a conserved β-barrel domain found in both SEBand SEA. However, the TSST-1 site lacks polarity equivalent to SEB/SEA,and hydrogen bonding with the hydroxyl of TSST-1 residue T69 wouldrequire that DRα K39 extend 5 Å into the pocket. TSST-1 binding utilizesan alternative strategy [Kim et al. (1994) Science 266:1870-1874]consisting of hydrophobic contacts centered around residue I46, andpotential ionic or hydrogen bonds bridging DRα residues E71 and K67 toR34 and D27, respectively, of TSST-1.

The hydrophobic region of the binding interface between SEB and theHLA-DR1 molecule consists of SEB residues 44-47, located in a largereverse turn connecting β-strands 1 and 2 of SEB. These residues appearto make strong electrostatic interactions with DRα through theirbackbone atoms. The mutation of L45 to an arginine reduced overallHLA-DR1 binding greater than 100-fold (FIG. 2b), attributable to theless energetically favorable insertion of a highly charged residue intoa hydrophobic depression on the DR1 molecule. The modeled DR1-SEAcomplex presents similar interactions with the SEA backbone atoms, withthe exception of a glutamine (Q49) replacing SEB Y46. Mutation of L48 toglycine in SEA (homologous to L45 of SEB) has been reported to decreaseT-cell responses. SEB L45 and the comparable L30 of TSST-1 are the mostextensively buried residues in the DR1 interface. The leucine isconserved among the bacterial superantigens (FIG. 3) and may provide thenecessary hydrophobic structural element for surface complementaritywith DR1, consistent with the mutagenesis data for SEB and SEA.

The inventor has performed similar structure and function studies withTSST-1, SEC1 and SPE-A.

In determining the overall affinity of the superantigen for DR1, acontributory role is played by structural variations around the commonbinding motifs. A short, variable structured, disulfide-bonded loop isfound in SEA and a homologous longer loop in SEB. The SEB residue Y94,contained within this loop, forms hydrophobic interactions with L60 andA61 of the DRα subunit. Replacement of Y94 with alanine partiallyinhibits DR1 binding (FIGS. 2a,b). An alanine is found in SEA (A97) andSEE at the position equivalent to SEB Y94, and mutating this residue inSEA to tyrosine results in disrupted instead of stabilized interactionswith DR1 (FIG. 2a). Although the disulfide loops differ in structurebetween SEA and SEB, A97 apparently contributes to the DRα bindinginterface in a manner similar to Y94 of SEB. Because TSST-1 lacks adisulfide loop, similar contacts with DRα are replaced by interactionswith β-strands of TSST-1. In a like manner, the absence of a salt bridgebetween the residues K39 of DRα and N65 of SED is apparently compensatedfor by stabilizing interactions occurring outside of the otherwiseconserved dominant binding surfaces (FIG. 2a).

The amino acid residues in contact with TCR are located in regions ofhigh sequence variability, presenting a unique surface for interactionwith the TCR. Residues implicated in TCR interactions by mutagenesis ofSEA and SEB reside in variable loop regions, while TSST-1 mutants thataffect TCR binding are mainly located in an a helix [Acharya, R. K. etal. (1994) Nature 367, 94-97; Kim, J. et al. (1994) Science 266,1870-1874]. Specifically, mutations that diminish T-cell receptorrecognition of SEB include residues N23, Y61, and the homologous SEA N25or Y64 (FIG. 2c). SEA residues S206 and N207 also control T-cellresponses [Hudson, et al. (1992) J. Exp. Med. 177: 175-184]. Mutants ofthe polar binding pocket, SEA Y92A and SEB Y89A, equivalently reducedT-cell responses (FIG. 2c), reflecting the observed decreases inDR1-binding (FIGS. 2a, b). While supporting reduced T-cell responses,mutants SEA Y64A and SEB Y61A retained normal affinities for DR1 (FIGS.2a-c).

In view of the detailed description of the present invention and theresults of molecular modelling and structural studies of staphylococcaland streptococcal superantigen toxins discussed above, any amino acidsequence derived from a superantigen toxin can be altered. Sequences ofseveral superantigen toxins are already known and available to thepublic in sequence databases such as GenBank, for example. Thesuperantigen toxin sequence is preferably altered at the hydrophobicloop or polar binding pocket depending on the superantigen.Alternatively, residues adjacent to the hydrophobic loop or polarbinding pocket that contact HLA-DR or residues at sites that canindirectly alter the structure of the hydrophobic loop or polar pocketcan be altered. The number of residues which can be altered can vary,preferably the number can be 1-2, more preferably 2-3, and mostpreferably 3-4, or more with the limitation being the ability to analyzeby computational methods the consequences of introducing such mutations.The residues which can be altered can be within 5 amino acid residues ofthe central Leucine of the hydrophobic loop (such as L45 of SEB), orwithin 5 residues of one of the amino acid residues of the polar bindingpocket that can contact HLA-DR, (such as E67, Y89, or Y115 of SEB), morepreferably, within 3 amino acid residues of the central Leucine of thehydrophobic loop (such as L45 of SEB), or within 3 residues of one ofthe amino acid residues of the polar pocket that can contact HLA-DR,(such as E67, Y89, or Y115 of SEB), and most preferably, the centralLeucine of the hydrophobic loop (such as L45 of SEB), or one of theamino acid residues of the polar binding pocket that can contact HLA-DR,(such as E67, Y89, or Y115 of SEB). The residues can be changed orsubstituted to alanine for minimal disruption of protein structure, morepreferably to a residue of opposite chemical characteristics, such ashydrophobic to hydrophilic, acidic to neutral amide, most preferably byintroduction of a residue with a large hydrated side chain such asArginine or Lysine. In addition, side chains of certain nonconservedreceptor-binding surfaces, can also be altered when designingsuperantigen toxins with low binding affinities. These residues caninclude Y94 of SEB and structurally equivalent residues of othersuperantigens, such as A97 of SEA, or any side chain within 5 residuesfrom these positions or any side chain in discontinuous positions(discontinuous positions are defined as amino acid residues that foldtogether to form part of a discrete three-dimensional structural unitbut are not present on the same secondary structural unit e.g. α helixor β-strand) such as disulfide-bonded side chains, that involve,directly or indirectly, the nonconserved receptor contact surfacesoutside of the polar binding pocket or hydrophobic loop. Further, aminoacid residues involved with protein folding or packing can be alteredwhen designing superantigen toxins with low binding affinities[Sundstrom et al. (1996) EMBO J. 15, 6832-6840; Sundstrom et al. (1996)J. Biol. Chem. 271, 32212-32216; Acharya et al. (1994) Nature 367,94-97; Prasad et al. (1993) Biochem. 32, 13761-13766; Swaminathan et al.(1992) Nature 359, 801-806]. Furthermore, especially for superantigenswith higher affinities for T-cell antigen receptors, side chains ofamino acids within 5 residues of the position represented by N23(conserved residue in most superantigens), N60 (conserved Asn or Trp inmost superantigens) Y91 (semiconserved hydrophobic residues Trp, Ile,Val, His in most superantigens) and D210 of SEB (conserved Asp in mostsuperantigens) can be altered when designing superantigen toxins withlow binding affinities. These residues are likely to form part of theintegral molecular surfaces that are in contact with T-cell antigenreceptors. Because the T-cell receptor contact areas of superantigentoxins are essential for causing specific activation or inactivation ofT-cell subsets, altering residues that are unique to each superantigenbut that are located within 5 residues of the positions represented byN23, N60 and Y91 can produce superantigens that affect a smaller number(e.g. 1-3) of subsets. Such altered superantigen toxins can be useful astherapeutic agents.

In another embodiment, the present invention relates to a DNA or cDNAsegment which encodes a superantigen toxin such as SEA corresponding tonucleotides 1 to 773 of SEQ ID NO:1 or nucleotides 1 to 700 of SEQ IDNO:3, SEB corresponding to nucleotides 244 to 1044 of SEQ ID NO:5, ornucleotides 244 to 1044 of SEQ ID NO:7, or nucleotides 1 to 720 of SEQID NO:9, SEC-1 corresponding to nucleotides 118 to 918 of SEQ ID NO:13,SPEa corresponding to nucleotides 814 to 1569 of SEQ ID NO:15, andTSST-1 corresponding to nucleotides 16 to 720 of SEQ ID NO:11 , to namea few, the sequence of which has been altered as described above toproduce a toxin protein with altered binding ability to MHC Class IIand/or T-cell receptors. For SEA, the following three mutations wereintroduced into the toxin molecule: Tyrosine at amino acid position 92changed to alanine; Aspartic acid at amino acid position 70 changed toarginine; Leucine at amino acid position 48 changed to arginine. Thereduction in binding to HLA DR is additive per mutation, though one ortwo mutations can produce a vaccine and a combination of all threemutations in one molecule produces a better vaccine. Other substitutionscan also result in reduced binding.

The B899445 vaccine consists of the following three mutationssimultaneously introduced into the toxin molecule: tyrosine at aminoacid position 89 changed to alanine; tyrosine at amino acid position 94changed to alanine; leucine at amino acid position 45 changed toarginine. The altered superantigen toxins can be expressed either as afull-length propolypeptide or as a polypeptide in which the leaderpeptide has been deleted. The full-length expressed product (SEAvaccine, A489270P (SEQ ID NO:12); SEB vaccine B899445P (SEQ ID NO:8),B2360210P (SEQ ID NO:6)) is secreted into the periplasmic space of E.coli host cells, and the leader peptide is recognized and cleaved by anative bacterial enzymatic mechanism. The altered superantigen toxins inwhich the leader peptide has been deleted (A489270C (SEQ ID NO:4),B899445C (SEQ ID NO:10)), the first residue of the mature protein isencoded by the transcriptional start site and codon for methionine(ATG), and the protein is expressed as a nonsecreted product within thehost E. coli cell. For the TSST-1 vaccine TST30 (SEQ ID NO:12), theleucine at position 30 was changed to arginine. For the SEC1 vaccine,SEC45 (SEQ ID NO:14), the leucine at position 45 was changed toarginine. For the SPE-A vaccine, SPEA42 (SEQ ID NO:16), the leucine atposition 42 was changed to arginine.

In another embodiment, the present invention relates to a recombinantDNA molecule that includes a vector and a DNA sequence as describedabove. The vector can take the form of a plasmid such as any broad hostrange expression vector for example pUC18/19, pSE380, PHIL, pET21/24 andothers known in the art. The DNA sequence is preferably functionallylinked to a promoter such that the gene is expressed when present in anexpression system and an altered superantigen toxin is produced. Theexpression system can be an in vitro expression system or host cellssuch as prokaryotic cells, or in vivo such as DNA vaccines.

In a further embodiment, the present invention relates to host cellsstably or transiently transformed or transfected with theabove-described recombinant DNA constructs. The host can be anyeukaryotic or prokaryotic cell including but not limited in E. coli DH5αor BL21. The vector containing the altered superantigen toxin gene isexpressed in the host cell and the product of the altered toxin gene,whether a secreted mature protein or a cytoplasmic product, can be usedas a vaccine or as a reagent in diagnostic assays or detection methods,or for therapeutic purposes. Please see e.g., Maniatis, Fitsch andSambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning,Volumes I and II (D. N. Glover ed. 1985) for general cloning methods.The DNA sequence can be present in the vector operably linked to ahighly purified IgG molecule, an adjuvant, a carrier, or an agent foraid in purification of altered toxin. The transformed or transfectedhost cells can be used as a source of DNA sequences described above.When the recombinant molecule takes the form of an expression system,the transformed or transfected cells can be used as a source of thealtered toxin described above.

A recombinant or derived altered superantigen toxin is not necessarilytranslated from a designated nucleic acid sequence; it may be generatedin any manner, including for example, chemical synthesis, or expressionof a recombinant expression system. In addition the altered toxin can befused to other proteins or polypeptides for directing transport forexample into the periplasm or for secretion from the cell. This includesfusion of the recombinant or derived altered superantigen to othervaccines or sequences designed to aid in purification, such asHis-tagged, epitope-tagged or antibody Fc-fusions.

In a further embodiment, the present invention relates to a method ofproducing altered superantigen toxin which includes culturing theabove-described host cells, under conditions such that the DNA fragmentis expressed and a superantigen toxin protein is produced. Thesuperantigen toxin can then be isolated and purified using methodologywell known in the art such as immunoaffinity chromatography orpreparative isoelectric focusing. However, the method of purification isnot critical to the performance of the vaccine. The altered superantigentoxin can be used as a vaccine for immunity against infection withbacterial superantigen toxins or as a diagnostic tool for detection ofsuperantigen toxin-associated disease or bacterial infection. Thetransformed host cells can be used to analyze the effectiveness of drugsand agents which affect the binding of superantigens to MHC class II orT-cell antigen receptors. Chemically derived agents, host proteins orother proteins which result in the down-regulation or alteration ofexpression of superantigen toxins or affect the binding affinity ofsuperantigen toxins to their receptors can be detected and analyzed. Amethod for testing the effectiveness of a drug or agent capable ofaltering the binding of superantigen toxins to their receptors can befor example computer-aided rational design or combinatorial libraryscreening, such as phage display technology.

In another embodiment, the present invention relates to antibodiesspecific for the above-described altered superantigen toxins. Forinstance, an antibody can be raised against the complete toxin oragainst a portion thereof. Persons with ordinary skill in the art usingstandard methodology can raise monoclonal and polyclonal antibodies tothe altered superantigens of the present invention, or a unique portionof the altered superantigen. Materials and methods for producingantibodies are well known in the art (see for example Goding, in,Monoclonal Antibodies: Principles and Practice, Chapter 4, 1986). Theantibodies can be used in diagnostic assays for detection ofsuperantigen toxin-associated infection. Neutralizing antibodies can beused in a therapeutic composition for the treatment of amelioration ofanergy and/or for the treatment of a superantigen toxin-associatedinfection.

In a further embodiment, the present invention relates to a method fordetecting the presence of superantigen-associated bacterial infectionsin a sample. Using standard methodology well known in the art, adiagnostic assay can be constructed by coating on a surface (i.e. asolid support) for example, a microtitration plate or a membrane (e.g.nitrocellulose membrane), all or a unique portion of the alteredsuperantigen described above, and contacting it with the serum of aperson suspected of having a superantigen-associated bacterialinfection. The presence of a resulting complex formed between thealtered superantigen toxin and antibodies specific therefor in the serumcan be detected by any of the known methods common in the art, such asfluorescent antibody spectroscopy or colorimetry. This method ofdetection can be used, for example, for the diagnosis ofsuperantigen-associated bacterial infections.

In yet another embodiment, the present invention relates to a method fordetecting the presence of superantigen toxin in a sample. Using standardmethodology well known in the art, a diagnostic assay can be constructedby coating on a surface (i.e. a solid support) for example, amicrotitration plate or a membrane (e.g. nitrocellulose membrane),antibodies specific for altered superantigen toxin, and contacting itwith serum or tissue sample of a person suspected of havingsuperantigen-associated bacterial infection. The presence of a resultingcomplex formed between toxin in the serum and antibodies specifictherefor can be detected by any of the known methods common in the art,such as fluorescent antibody spectroscopy or colorimetry. This method ofdetection can be used, for example, for the diagnosis ofsuperantigen-associated bacterial infection or disease such as foodpoisoning and toxic-shock syndrome or the detection of superantigentoxin in food and drink.

In another embodiment, the present invention relates to a diagnostic kitwhich contains altered superantigen toxin from a specific bacteria orseveral different superantigen toxins from bacteria and ancillaryreagents that are well known in the art and that are suitable for use indetecting the presence of antibodies to superantigen toxin-associatedbacteria in serum or a tissue sample. Tissue samples contemplated can beavian, fish, or mammal including monkey and human.

In yet another embodiment, the present invention relates to a vaccinefor protection against superantigen toxin-associated bacterialinfections. The vaccine can comprise one or a mixture of individualaltered superantigen toxins, or a portion thereof. When a mixture of twoor more different altered superantigen toxin from different bacteria isused, the vaccine is referred to as a multivalent bacterial superantigenvaccine. The vaccine is designed to protect against the pathologiesresulting from exposure to one or several related staphylococcal andstreptococcal toxins. In addition, the protein or polypeptide can befused or absorbed to other proteins or polypeptides which increase itsantigenicity, thereby producing higher titers of neutralizing antibodywhen used as a vaccine. Examples of such proteins or polypeptidesinclude any adjuvants or carriers safe for human use, such as aluminumhydroxide.

The staphylococcal enterotoxin (SE) serotypes SEA, SED, and SEE areclosely related by amino acid sequence, while SEB, SEC1, SEC2, SEC3, andthe streptococcal pyrogenic exotoxins B share key amino acid residueswith the other toxins, but exhibit only weak sequence homology overall.However, there are considerable similarities in the knownthree-dimensional structures of SEA, SEB, SEC1, SEC3, and TSST-1.Because of this structural similarity, it is likely that polyclonalantibodies obtained from mice immunized with each SE or TSST-1 exhibit alow to high degree of cross-reaction. In the mouse, these antibodycross-reactions are sufficient to neutralize the toxicity of most otherSE/TSST-1, depending upon the challenge dose. For example, immunizationwith a mixture of SEA, SEB, TSST-1 and SPEa was sufficient to provideantibody protection from a challenge with any of the component toxins,singly or in combination.

The likelihood of substantial antigen-cross-reactivity suggests that itmay be possible to obtain immune protection for other (or perhaps all)staphylococcal superantigens by use of a minimal mixed composition ofvaccines. For the case of staphylococcal superantigens, a combination ofthe component vaccines from SEA, SEB, SEC-1 and TSST-1 should besufficient to provide immune protection against SEA, SEB, SEC1-3, andTSST-1. The addition of SPEa component to the trivalent mixture willallow for sufficient protection against the streptococcal toxins SPEaand SPEc. Therefore, a multivalent vaccine consisting of the alteredsuperantigen toxins from SEA, SEB, SEC-1, TSST-1, and SPEa as describedabove, is predicted to provide protective immunity against the majorityof bacterial superantigen toxins.

The vaccine can be prepared by inducing expression of a recombinantexpression vector comprising the gene for the altered toxin describedabove. The purified solution is prepared for administration to mammalsby methods known in the art, which can include filtering to sterilizethe solution, diluting the solution, adding an adjuvant and stabilizingthe solution. The vaccine can be lyophilized to produce a vaccineagainst superantigen toxin-associated bacteria in a dried form for easein transportation and storage. Further, the vaccine may be prepared inthe form of a mixed vaccine which contains the altered superantigentoxin(s) described above and at least one other antigen as long as theadded antigen does not interfere with the effectiveness of the vaccineand the side effects and adverse reactions, if any, are not increasedadditively or synergistically. Furthermore, the vaccine may beadministered by a bacterial delivery system and displayed by arecombinant host cell such as Salmonella spp, Shigella spp,Streptococcus spp. Methods for introducing recombinant vectors into hostcells and introducing host cells as a DNA delivery system are known inthe art [Harokopakis et al. (1997) Infect. Immun. 65, 1445-1454;Anderson et al. (1996) Vaccine 14, 1384-1390; Medaglini et al. (1995)Proc. Natl. Acad. Sci. U.S.A. 92, 6868-6872].

The vaccine may be stored in a sealed vial, ampule or the like. Thepresent vaccine can generally be administered in the form of a liquid orsuspension. In the case where the vaccine is in a dried form, thevaccine is dissolved or suspended in sterilized distilled water beforeadministration. Generally, the vaccine may be administered orally,subcutaneously, intradermally or intramuscularly but preferablyintranasally in a dose effective for the production of neutralizingantibody and protection from infection or disease.

In another embodiment, the present invention relates to a method ofreducing superantigen-associated bacterial infection symptoms in apatient by administering to said patient an effective amount ofanti-altered superantigen toxin antibodies, as described above. Whenproviding a patient with anti-superantigen toxin antibodies, or agentscapable of inhibiting superantigen function to a recipient patient, thedosage of administered agent will vary depending upon such factors asthe patient's age, weight, height, sex, general medical condition,previous medical history, etc. In general, it is desirable to providethe recipient with a dosage of the above compounds which is in the rangeof from about 1 pg/kg to 10 mg/kg (body weight of patient), although alower or higher dosage may be administered.

In a further embodiment, the present invention relates to a therapeuticmethod for the treatment of diseases that may not be associated directlywith superantigen toxins but which result in specific nonresponsivenessof T-cell subsets or detection of abnormally low level of subsets inperipheral blood, said method comprising the administration of alteredsuperantigen toxins, in vivo or ex vivo, such that T-cell subsets areexpanded or stimulated. Diseases which cause anergy or nonresponsivenessof T-cells include, but are not limited to, infectious diseases andcancers. The desired clinical outcome such as an increase in detectableT cell subsets or in stimulation ex vivo of T-cells through theirantigen receptors, such as by antigen or anti-CD3 antibody can bemeasured by standard clinical immunology laboratory assays.

In yet another embodiment, the present invention relates to atherapeutic method for the treatment of diseases associated withexpanded or over-stimulated T-cell subsets, such as autoimmunity forexample, said method comprising administration in vivo or ex vivo, ofsuperantigen toxin altered in such a manner that only limited (1-3)T-cell subsets are stimulated but that MHC class II binding affinitystill remains, such that anergy or inactivation of T-cells is produced.The desired clinical outcome can be measured as a reduction ofcirculating blood T-cells of the targeted subset(s) or diminishedantigen or other antigen receptor-mediated-stimulatory responses byassays known in the art.

Described below are examples of the present invention which are providedonly for illustrative purposes, and not to limit the scope of thepresent invention. In light of the present disclosure, numerousembodiments within the scopy of the claims will be apparent to those ofordinary skill in the art.

The following Materials and Methods were used in the Examples thatfollow.

Structural Comparisons

Primary protein structure data are available for several bacterialsuperantigens, including SEA, SED, SEB, SEC1-3, TSST-1. Superantigensfor which structures were unavailable were modeled using comparativetechniques (HOMOLOGY program; Biosym Technologies, Inc., San Diego,Calif.). Before x-ray crystallography data was available, SEA wasmodeled by using this method, and the model was in very close agreementwith the experimentally determined structure. As an example, the aminoacid sequence for SEA was aligned with the known structure of free andHLA-DR1 bound SEB, and the SEA molecule was built for both free andDR1-bound proteins. Loop segments of SEA were generated by a de novomethod. Refinement of the modeled structures was carried out by means ofmolecular-dynamics simulations (DISCOVER, Biosym). The constructed freeSEA molecule was immersed in a 5-Å layer of solvent water and theα-carbon atoms lying in the structurally conserved regions were tetheredto their initial positions during the simulations. For the bound SEAmolecule, simulations were carried out by constructing an active-siteregion composed of part of the SEA molecule and the DR1 molecule insidea 10-Å interface boundary, as derived from the crystal structure of theDR1-SEB complex. Amino acid residues lying in the outer boundary wererigidly restrained at their initial positions. The active-site regionwas immersed in a 5-Å layer of water. Protein interactions were modeledby employing the consistent valence force field with a non-bonded cutoffdistance of 11.0 Å. Simulations were initiated with 100 cycles ofminimization using a steepest descent algorithm followed by 100-psrelaxation (using a 1.0 fs timestep). Structural comparisons betweenSEB, SEC1, and TSST-1 were performed by using the crystal structures(Brookhaven data holdings) aligned according to common secondarystructural elements and/or by sequence and structural homology modeling.

Site-specific Mutagenesis

Site-specific mutagenesis was performed according to the methoddeveloped by Kunkel, using gene templates isolated from Staphylococcusaureus strains expressing SEA (FDA196E, a clinical isolate, Fraser, J.D. (1994) Nature 368: 711-718), SEB (14458, clinical isolate), SEC1(Toxin Technologies, Sarasota, Fla.), TSST-1 (pRN6550 cloned product, aclinical isolate, Kreiswirth, B. N. et al. (1987) Mol. Gen. Genet. 208,84-87), and SPEa (Toxin Technologies), respectively. Modified T7polymerase (Sequenase, U.S. Biochemical Corp., Cleveland, Ohio) was usedto synthesize second-strand DNA from synthetic oligonucleotidesharboring the altered codon and single-stranded, uracil-enriched M13templates. Mutagenized DNA was selected by transforming E. coli strainJM101. Alternatively, double stranded DNA was used as template formutagenesis. Mutagenized sequences were confirmed by DNA sequencing(Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463-5467; Sambrooket al., 1989) using synthetic primers derived from known sequences, oruniversal primers. The complete coding sequences were inserted intoexpression plasmids such as pUC19, pSE380 or pET21 for production in E.coli hosts.

Protein Purifications

The appropriate E. coli hosts were transformed with plasmids pETA489270C(ATCC 98449, deposited at American Tissue Culture Collection, Manassas,Va. under the terms of the Budapest Treaty on Jun. 4, 1997), pETB2360210(ATCC 98447, deposited at ATCC under the terms of the Budapest Treaty onJun. 4, 1997), pETB899445P (ATCC 98446, deposited at ATCC under theterms of the Budapest Treaty on Jun. 4, 1997) harboring the mutant toxingenes. In general, the bacteria were grown to an A600 0.5-0.6 inTerrific Broth (Difco Laboratories, Detroit, Mich.) containing 50 μg/mLampicillin or kanamycin. Recombinant proteins were induced withisopropyl-β-D-thio-galactopyranoside (Life Technologies, Gaithersburg,Md.) and recovered as cytoplasmic or bacterial periplasmic secretionproducts. Bacteria were collected by centrifugation, washed with 30 mMNaCl, 10 mM TRIS (pH 7.6), and pelleted by centrifugation and eitherlysed or osmotically shocked for collection of secreted proteins.Preparations were isolated by CM Sepharose ion-exchange chromatography,rabbit antibody (Toxin Technologies, Sarasota, Fla.) affinity columns,ion exchange HPLC or similar methods. In some cases partially purifiedsuperantigen was further purified by preparative isoelectric focusing(MinipHor; Rainin Instrument Company, Inc., Woburn, Mass.). The MinipHorwas loaded with the SEA-enriched fraction from CM Sepharosechromatography in a solution containing 10% (v/v) glycerol and 1% (v/v)pH 6-8 ampholytes (Protein Technologies, Inc., Tucson, Ariz.). Theprotein preparations were allowed to focus until equilibrium was reached(approximately 4 hr, 4° C.). Twenty focused fractions were collected andaliquots of each were analyzed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblotting. The SEA-containing fractions were pooled,and refocused for an additional 4 h. The fractions containing purifiedSEA were pooled and dialyzed first against 1 M NaCl (48 h, 4° C.) toremove ampholytes, and then against PBS (12 h, 4° C.). Legitimateamino-terminal residues were confirmed by protein sequencing. Precisemeasurements of protein concentrations were performed by immunoassayusing rabbit antibody affinity-purified with the wild-type superantigensand by the bicinchoninic acid method (Pierce, Rockford, Ill.) usingwild-type protein as standards. All protein preparations were >99% pure,as judged by SDS-PAGE and Western immunoblots. In some cases, as whenused for lymphocyte assays, bacterial pyrogens were removed by passingthe protein preparations over Polymyxin B affinity columns.

Binding of Superantigens to HLA-DR1

The DR1 homozygous, human B-lymphoblastoid cell line LG2 or L cellstransfected with plasmids encoding HLA-DR1αβ were used in the bindingexperiments. Cells were incubated 40 min (37° C.) with wild-type ormutant superantigen in Hanks balanced salt solution (HBSS) containing0.5% bovine serum albumin. The cells were washed with HBSS and thenincubated with 5 μg of specific rabbit antibody (Toxin Technology,Sarasota, Fla.) for 1 h on ice. Unbound antibody was removed, and thecells were incubated with FITC-labelled goat anti-rabbit IgG (OrganonTeknika Corp., Durham, N.C.) on ice for 30 min. The cells were washedand analyzed by flow cytometry (FACScan; Becton Dikinson & Co., MountainView, Calif.). Controls consisted of cells incubated with affinitypurified anti-toxin and the FITC labelled antibody without prioraddition of superantigen.

Lymphocyte Proliferation

Human peripheral blood mononuclear cells were purified by Ficoll-hypaque(Sigma, St. Louis, Mo.) buoyant density gradient centrifugation. Genesencoding the human MHC class II molecules DR1αβ (DRA and DRB1*0101 cDNA[Bavari and Ulrich (1995) Infect. Immun. 63, 423-429] were cloned intothe eukaryotic expression vector pRC/RSV (Invitrogen, Carlsbad, Calif.),and mouse L cells were stably transfected. The transfectants wereselected by fluorescence-activated cell sorting (EPICS C, Coulter Corp.,Hialeah, Fla.) using rabbit anti-DRαβ antisera and FITC-goat anti-rabbitIgG, to produce cells that expressed a high level of DRαβ21. 1×10⁵cells/well of a 96-well plate were irradiated (15,000 Rad), andwild-type or mutant SE, was added. After a brief incubation period (45min, 37° C.), unbound SE was rinsed from the culture plates using warmmedia. The cells were cultured in RPMI-1640 (USAMRIID) with 5% FBS for72 h, and pulsed-labelled for 12 h with 1 μCi [³H]-thymidine (Amersham,Arlington Heights, Ill.). Cells were harvested onto glass fiber filters,and [³H]-thymidine incorporation into the cellular DNA was measured by aliquid scintillation counter (BetaPlate, Wallac Inc., Gaithersburg,Md.). Splenic mononuclear cells or human peripheral blood mononuclearcells were obtained by buoyant density centrifugation (Histopaque; SigmaChemical Comp.) and washed three times. The cells were resuspended inmedium containing 5% fetal bovine serum (FBS), and 100 μl (4×10⁵ cells)of the cell suspension was added to triplicate wells of 96-well flatbottom plates. The mononuclear cells were cultured (37° C., 5% CO₂) withWT or mutant SEA. After 3 days the cultures were pulsed (12 h) with 1μCi/well of [³H]thymidine (Amersham, Arlington Heights, Ill.) andincorporated radioactivity was measured by liquid scintillation.

Gel Electrophoresis and Immunoblotting Analysis.

The protein preparations were analyzed by SDS-PAGE (12%) and stainedwith Coomassie Brilliant Blue R-250 (Sigma Chemical Comp. St Louis, Mo.)in methanol (10% v/v) acetic acid (10% v/v). The proteins separated bySDS-PAGE (not stained) were transferred to nitrocellulose membranes(Bio-Rad Lab. Inc., Melville, N.Y.) by electroblotting, and themembranes were then blocked (12 h, 4° C.) with 0.2% casein in a bufferconsisting of 50 mM sodium phosphate, 140 mM sodium chloride, pH 7.4(PBS). The membrane was then incubated (1 h, 37° C., shaking) with 2μg/mL of affinity-purified anti-toxin antibody (Toxin Technology,Sarasota, Fla.) in PBS with 0.02% casein. After the membranes werethoroughly washed, peroxidase-conjugated goat anti-rabbit IgG(Cappel/Organon Teknika Corp., West Chester, Pa.) was added (1:5,000)and the membranes were incubated for 1 h (37° C.) with shaking. Theunbound antibody was removed by washing with PBS and bound antibody wasvisualized by using a Bio-Rad peroxidase development kit (Biorad,Hercules, Calif.). For quantitation, dilutions of wild-type preparationswere immobilized on nitrocellulose membranes by using a Slot-Blotapparatus (Bio-Rad). The membrane was removed from the Slot-Blotapparatus and unreacted sites were blocked (12 h, 4° C.) with 0.2%casein in PBS. After washing once with the PBS, the membrane wasincubated (1 h, 37° C.) with 2 μg/mL rabbit affinity purified anti-toxinantibody (Toxin Technology) in PBS that contained 0.02% casein. Afterfour washes, the bound rabbit antibody was reacted with goat anti-rabbitIgG conjugated with horseradish peroxidase (1 h, 37° C.) and the blotswere developed using enhanced chemiluminescence (ECL; Amersham LifeSciences, Arlington Heights, Ill.) or similar methods. The amount ofmutant protein was measured by densitometry (NIH Image 1.57 software,National Institutes of Health, Bethesda, Md.) of exposed X-ray film.Standard curves were prepared by plotting the mean of duplicatedensitometric readings for each dilution of toxin standard. Theresulting values were fitted to a straight line by linear regression.Concentrations of proteins were determined by comparing mean values ofvarious dilutions of the mutant to the standard curve.

Biological Activities and Immunizations.

Male C57BL/6 mice, 10 to 12-weeks old, were obtained from HarlanSprague-Dawley, Inc. (Frederick Cancer Research and Development Center,Frederick, Md.). The lethal effect of WT or mutant SEA was evaluated asdescribed in Stiles et al. (1993) Infect. Immun. 61, 5333-5338. Forimmunizations, mice were given by interperitoneal (ip) injections either2 or 10 μg of WT or mutant toxin in 100 μl of adjuvant (RIBI, ImmunochemResearch, Inc. Hamilton, Mont. or alum), or adjuvant only, and boosted(ip) at 2 and 4 weeks. Serum was collected from tail veins one weekafter the last immunization. Mice were challenged 2 weeks after the lastinjection with toxin and lipopolysaccharide (LPS, 150 μg) from E. coli055:B5 serotype (Difco Laboratories, Detroit, Mich.). Challenge controlswere adjuvant-immunized or non-immunized mice injected with both agents(100% lethality) or with either wild type toxin or LPS. No lethality wasproduced by these negative controls. Monkeys were immunized with theantigen in the right leg, caudal thigh muscles. Each received threeintramuscular immunizations with a superantigen vaccine plus adjuvant.Control monkeys received 0.5 ml total volume of adjuvant (Alhydrogel,Michigan Department of Public Health) and sterile PBS using the sametechniques and equipment as the immunized monkeys. Immunizations wereadministered 28±2 days apart and consisted of 20 μg of the vaccine inadjuvant in a total volume of 0.5 ml. Immunizations were administered onday 0, 28±2, and 56±2 using a 23-27 ga ½-⅝″ needle attached to a 1 mltuberculin syringe into the caudal thigh.

Antibody Assay.

Microtiter plates were coated with 1 μg/well of WT toxin in 100 μl ofPBS (37° C., 2 h). After antigen coating, the wells were blocked with250 μl of casein 0.2% in PBS for 4 h at 37° C. and then washed fourtimes with PBS containing 0.2% Tween 20. Immune or nonimmune sera werediluted in PBS containing 0.02% casein and 100 μl of each dilution wasadded to duplicate wells. After each well was washed four times, boundantibody was detected with horse radish peroxidase (Sigma ChemicalComp., St. Louis, Mo.) labelled goat anti-species specific IgG (37° C.,1 h), using O-phenylenediamine as the chromogen. Mean of duplicates OD(absorbance at 490 nm) of each treatment group was obtained and thesedata were compared on the basis of the inverse of the highest serumdilution that produced an OD reading four times above the negativecontrol wells. For negative controls, antigen or serum was omitted fromthe wells.

Superantigen Binding and TCR Subset Analysis.

Cells from the mouse B-lymphoma line A20 (ATCC, Rockville, Md.) (2-4×10⁵cells) were incubated (40 min at 37° C.) with WT or mutant toxin inHanks balanced salt solution containing 0.5% bovine serum albumin (HBSS,USAMRIID). The cells were washed with HBSS and incubated with 5 μg ofaffinity-purified anti-toxin antibody in HBSS (4° C., 45 min). Unboundantibody was removed and the bound antibody was detected withfluorescein isothiocyanate (FITC)-labelled, goat anti-rabbit IgG(Organon Teknika Corp., Durham, N.C.). Unbound antibody was removed andthe cells were analyzed by with a FACSort flow cytometer (BectonDikinson & Co., Mountain View, Calif.).

For TCR subset analysis, splenic mononuclear cells were obtained frommice immunized with WT or mutant toxin. The mononuclear cells wereincubated (37° C.) with WT toxin (100 ng/mL) for 5 days and thencultured in 85% RPMI-1640, 10% interleukin-2 supplement (AdvancedBiotechnologies Inc., Columbia, Md.) with 5% FBS for an additional 5days. The T cells were washed twice and stained with anti-TCR(Biosource, Camarillo, Calif.) or anti-Vβ specific TCR (Biosource,Camarillo, Calif.) (45 min, 4° C.). All cells analyzed were positive forT cell marker CD3+ and expressed the CD25 activation marker (data notshown). Controls were incubated with an isotype matched antibody ofirrelevant specificity. Unreacted antibody was removed, and the cellswere incubated with an FITC-labelled, anti-mouse IgG (Organon TeknikaCorp, Durham, N.C.) on ice for 30 min. The cells were washed andanalyzed by flow cytometry (FACSort).

LPS Potentiation of SE Toxicity in Mice.

C57BL/6 or BALB/c mice weighing 18-20 g (Harlan Sprague Dawley, Inc.,Frederick Cancer Research and Development Center, Frederick, Md.) wereeach injected intraperitoneally (i.p.) with 200 μl of PBS containingvarying amounts of SEA, SEB, or SEC1, TSST-1, or SPEa followed 4 h laterwith 75 or 150 μg of LPS (200 μl/i.p.). Controls were each injected witheither SE (30 mg) or LPS (150 mg). Animals were observed for 72 h afterthe LPS injection. Calculations of LD50 were done by Probit analysisusing 95% fiducial limits (SAS Institute Inc., Cary, N.C.).

The biological effects of SEA and SEB were also tested in transgenicC57BL/6 mice (GenPharm International, Mountain View, Calif.) deficientin MHC class I or II expression [Stiles et al. (1993) Infect. Immun. 61,5333-5338], as described above, using a single dose of toxin (30μg/mouse). Genetic homozygozity was confirmed by Southern analysis ofparental tail DNA, using β2 microglobulin and MHC class II β DNA probes.

Detection of Cytokines in Serum.

Mice (n=18 per group) were injected with toxin (10 μg), LPS (150 μg), ortoxin plus LPS. Sera were collected and pooled from three mice per groupat each time point (2, 4, 6, 8, 10, 22 h) after LPS injection. Sera werecollected at various time points following toxin injection (−4 h, or 4hbefore LPS injection, for data tabulation). Collection of LPS controlsera began at the time of injection (0 h).

Serum levels of TNFα and IL-α were detected by an enzyme linkedimmunosorbent assay (ELISA). TNFα was first captured by a monoclonalantibody against mouse TNFα (GIBCO-BRL, Grand Island, N.Y.) and thenincubated with rabbit anti-mouse TNFα antibody (Genzyme, Boston, Mass.).The ELISA plate was washed and peroxidase conjugate of anti-rabbitantibody (Boehringer Mannheim, Indianapolis, Ind.) added to the wells.After washing the plate and adding substrate (Kirkegaard and Perry,Gaithersburg, Md.), TNFα concentrations were measured using the meanA450 reading of duplicate samples and a standard curve generated fromrecombinant mouse TNFα (GIBCO-BRL). Serum levels of IL-1α weredetermined from the mean reading of duplicate samples with an ELISA kitthat specifically detects murine IL-1α (Genzyme, Boston, Mass.). Thestandard error of the mean (SEM) for TNFα and IL-1α readings was +/−5%.

Quantitation of IL-6 and IFNγ were measured by bioassays [See etal.(1990) Infect. Immun. 58: 2392-2396]. An IL-6 dependent cell line,7TD1 (kindly provided by T. Krakauer), was used in a proliferative assaywith serial two-fold dilutions of serum samples assayed in triplicate.Proliferation of 7TD1 cells in a microtiter plate was measured by uptakeof [³H]-thymidine (1 μCi/well; Amersham, Arlington Heights, Ill.) andthe activity of IL-6 from serum was compared to a recombinant mouse IL-6standard (R and D Systems, Minneapolis, Minn.) as previously described[See et al. (1990) Infect. Immun. 58: 2392-2396]. The SEM of triplicatesamples was +/−10%.

IFNγ was measured by the reduction of vesicular stomatitis virus (NewJersey strain) cytopathic effects on L929 cells, as previously described[Torre et al. (1993) J. Infect. Dis. 167, 762-765]. Briefly, serialtwo-fold dilutions of serum were made in duplicate and added tomicrotiter wells containing L929 cells (5×10⁴/well). After incubating 24h, virus (5×10⁵ PFU/well) was added and the cytopathic effects measuredat 48 h by absorbance readings (570 nm) of reduced3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (Sigma).The activity of each serum sample was determined using recombinant mouseIFNγ as a standard (Biosource, Camarillo, Calif.). The SEM of duplicatesamples was +/−5%.

EXAMPLE 1

Molecular Modelling and Structural Studies of Staphylococcal andStreptococcal Superantigens: Bacterial Superantigens Share Common3-dimensional Structure.

Comparison of amino acid sequences (FIG. 1) suggested that bacterialsuperantigens fall into groups consisting of (1) SEA, SED and SEE, (2)SEB, staphylococcal enterotoxins C1-C3 (SEC1-3), the streptococcalpyrogenic exotoxins A (SPE-A) and C (SPE-C), (3) TSST-1 and (4) theexfoliative toxins (ETA, ETB) and streptococcal pyrogenic exotoxin B(SPE-B), which are the most distant from the others in sequence.Although not available to the inventor when the inventions were firstconceived and proof of principle was obtained, the x-raycrystallographic structures of several bacterial superantigens are nowknown. Diverse superantigens, such as SEB and TSST-1, appear to havelittle sequence in common, yet they exhibit homologous protein foldscomposed largely of β strands [Prasad, G. S. et al. (1993) Biochemistry32, 13761-13766; Acharya, R. K. et al. (1994) Nature 367, 94-97;Swaminathan, S. et al. (1992) Nature 359, 801-806] within two distinctdomains. Differences between the proteins are located primarily inhighly variable regions comprised of several surface loops, such as thedisulfide-bonded loop which is absent from TSST-1 and at the aminoterminus.

The X-ray crystal structures of SEB and TSST-1 complexed with HLA DR1are known [Kim, J. et al. (1994) Science 266, 1870-1874; Jardetzky, T.S. et al. (1994) Nature 368, 711-718] and this data was useful to fullyexplain our results concerning attenuation of the superantigens bysite-specific mutagenesis. The region of HLA DR1 that contacts SEBconsists exclusively of α subunit surfaces. The main regions of SEBinvolved are two conserved sites: a polar pocket derived from three βstrands of the β barrel domain and a highly solvent-exposed hydrophobicreverse turn. The polar binding pocket of SEB contains a glutamate andtwo tyrosines that accommodate Lys39 of the α subunit of HLA DR1, whilethe hydrophobic region consists of a leucine and flanking residues thatmake several contacts with the HLA DRα chain. The HLA DR1 binding sitesof both TSST-1 and SEB overlap significantly. The hydrophobic bindingcontacts of other SAg with the HLA DRα chain have been proposed [Ulrichet al. (1995) Nature, Struct. Biol. 2, 554-560] to be similar to thosefound in SEB and TSST-1. A motif consisting of a leucine in a reverseturn [Ulrich et al. (1995), supra] is conserved among bacterialsuperantigens and may provide the key determinant (hydrophobic orotherwise) for binding HLA-DR. However, TSST-1 does not have a highlycharged residue in the polar pocket that interacts with Lys39 of the HLADRα chain and uses an alternative conformational binding mode thatallows TSST-1 to interact with HLA DR1 β-chain residues and thecarboxy-terminal region of the antigenic peptide.

Both SEA and SEE bind to the β subunit of DR by means of a single zincatom [Fraser, J. D. et al. (1992) Proc. Natl. Acad. Sci. USA 89,5507-5511]. The amino-terminal domain of SEA interfaces with the HLA DRαchain [Ulrich et al. (1995), supra], while SEA C-terminal domainresidues His187, His225 and Asp227 form a zinc-coordination complex,likely with His-81 from the β chain of an adjoining HLA DR molecule.However, our results have shown that binding of superantigen to the HLADRβ subunit does not directly stimulate T cells [Ulrich et al. (1995),supra] but increases the potential of the bound SEA to interact with theα chain of another HLA DR, thus increasing the biological potency.

EXAMPLE 2

Molecular Modelling and Structural Studies of Staphylococcal andStreptococcal Superantigens: A Detailed Protein Structure Analysis ofSEB and SEA Suggested that all Bacterial Superantigens have a CommonMechanism for Binding MHC Class II Receptors.

A least-squares superimposition of the unbound molecules of modeled SEAand the crystal structure of SEB, aligned according to theirstructurally conserved α-helical and β-strand regions, exhibited aglobal folding pattern which is very similar. Differences between thetwo structures are calculated to be located primarily in loops of lowsequence homologies, with the largest positional deviations occurringbetween structurally conserved regions of residues 18-20, 30-32,173-181, 191-194, and the cysteine-loop region (90-111). Only one ofthese regions in SEB makes significant contact (residue Y94 inparticular) with the HLA-DR1 molecule [Jardetzky, T. S. et al. (1994)Nature 368, 711-718].

The binding interface between SEB and HLA-DR1 consists principally oftwo structurally conserved surfaces located in the N-terminal domain: apolar binding pocket derived from three β-strand elements of theβ-barrel domain and a hydrophobic reverse turn. The binding pocket ofSEB contains residues E67, Y89 and Y115, and binds K39 of the DRαsubunit. For SEA, the binding interface with the DR molecule is modeledto contain a similar binding pocket consisting of residues D70, Y92 andY108. Mutation of residue Y89 in SEB or Y92 in SEA to alanine (FIG. 2)resulted in 100-fold reduction in DR1 binding. The substitution ofalanine for Y89 in SEB and Y92 in SEA eliminates the hydrogen bond withK39 and disrupts packing interactions with adjacent protein residues.Modeling of the SEA mutant Y92A predicts an increase insolvent-accessible surface area for Y108 by a factor of two greater thanthe wild-type structure, allowing the formation of a hydrogen bond tothe carboxylate group of D70 and thus disrupting key anchoring andrecognition points for HLA-DR1. This effect is expected to be somewhatless in SEB due to the longer side chain at E67. Substitution of SEBY115 with alanine also resulted in 100-fold reduction of binding. Incontrast, the same replacement of Y108 in SEA yielded little to nochange in DR1 binding (FIG. 2a), suggesting the primary importance ofSEA residues Y92 and D70 for stabilizing interactions with K39. The K39side chain of DRα forms a strong ion-pair interaction with the SEB E67carboxylate group and hydrogen bonds with the hydroxyl groups of Y89 andY115. Substitution of SEB E67 by glutamine reduced binding affinity by100-fold (FIG. 2), reflecting the replacement of the strong ionic bondwith a weaker hydrogen bond. To optimize ion-pair interactions of theanalogous SEA site, the shorter carboxylate side chain of D70 ispredicted to shift K39 of DRα, weakening interactions with SEA Y108. Thesubstitution of alanine for SEA Y108 is thus more easily accommodatedthan the homologous substitution of SEB Y115, without loss in DR1binding.

Comparisons of the polar pocket with other bacterial superantigens werethen made. SEC1-3 and SPE-A have conserved the critical DR1binding-interface residues (FIG. 1), and share with SEE and SEAsecondary structural elements of the DR1-binding surfaces. Asparagine inSED (N70) replaces the acidic side chain present in SEA, SEE, SPE-A andSEC1-3. Accordingly, for SED the salt bridge of the polar pocket islikely to be replaced by a hydrogen bond. Overall DR1 affinities for SEDand SEA appeared to be equivalent (FIG. 2b), indicating that otherinteractions may compensate for the absence in SED of the ion-pair foundin the other superantigens. For the case of TSST-1, mutating DRαresidues K39 to serine or M36 to isoleucine has been shown to greatlyreduce binding [Panina-Bordignon et al. (1992) J. Exp. Med. 176:1779-1784]. Although primarily hydrophobic, the critical TSST-1structural elements are conserved with the SEA and SEE polar bindingpocket. SEE residues Y89 and Y115 are homologous to T69 and 185 inTSST-1, respectively, and SEB E67 is replaced by I46. These TSST-1residues are positioned in a conserved β-barrel domain found in both SEEand SEA. However, the TSST-1 site lacks polarity equivalent to SEB/SEA,and hydrogen bonding with the hydroxyl of TSST-1 residue T69 wouldrequire that DRα K39 extend 5 Å into the pocket. TSST-1 binding utilizesan alternative strategy [Kim et al. (1994) Science 266: 1870-1874]consisting of hydrophobic contacts centered around residue I46, andpotential ionic or hydrogen bonds bridging DRα residues E71 and K67 toR34 and D27, respectively, of TSST-1.

The hydrophobic region of the binding interface between SEE and theHLA-DR1 molecule consists of SEE residues 44-47, located in a largereverse turn connecting β-strands 1 and 2 of SEB. These residues appearto make strong electrostatic interactions with DRα through theirbackbone atoms. The mutation of L45 to an arginine reduced overallHLA-DR1 binding greater than 100-fold (FIG. 2b), attributable to theless energetically favorable insertion of a highly charged residue intoa hydrophobic depression on the DR1 molecule. The modeled DR1-SEAcomplex presents similar interactions with the SEA backbone atoms, withthe exception of a glutamine (Q49) replacing SEB Y46. Mutation of L48 toglycine in SEA (homologous to L45 of SEB) has been reported to decreaseT-cell responses. SEB L45 and the comparable L30 of TSST-1 are the mostextensively buried residues in the DR1 interface. The leucine isconserved among the bacterial superantigens (FIG. 3) and may provide thenecessary hydrophobic structural element for surface complementaritywith DR1, consistent with the mutagenesis data for SEB and SEA.

The inventor has performed similar structure and function studies withTSST-1, SEC1 and SPE-A.

EXAMPLE 3

Molecular Modelling and Structural Studies of Staphylococcal andStreptococcal Superantigens: Some Interactions of BacterialSuperantigens with MHC Class II Receptors are Not Conserved but are LessImportant than the Hydrophobic Loop and Polar Pocket Binding Sites.

In determining the overall affinity of the superantigen for DR1, acontributory role is played by structural variations around the commonbinding motifs. A short, variable structured, disulfide-bonded loop isfound in SEA and a homologous longer loop in SEB. The SEB residue Y94,contained within this loop, forms hydrophobic interactions with L60 andA61 of the Dα subunit. Replacement of Y94 with alanine partiallyinhibits DR1 binding (FIGS. 2a,b). An alanine is found in SEA (A97) andSEE at the position equivalent to SEB Y94, and mutating this residue inSEA to tyrosine results in disrupted instead of stabilized interactionswith DR1 (FIG. 2a). Although the disulfide loops differ in structurebetween SEA and SEB, A97 apparently contributes to the DRα bindinginterface in a manner similar to Y94 of SEB. Because TSST-1 lacks adisulfide loop, similar contacts with DRα are replaced by interactionswith β-strands of TSST-1. In a like manner, the absence of a salt bridgebetween the residues K39 of DRα and E67 of SED is apparently compensatedfor by stabilizing interactions occurring outside of the otherwiseconserved dominant binding surfaces (FIG. 2a).

EXAMPLE 4

Molecular Modelling and Structural Studies of Staphylococcal andStreptococcal Superantigens: Superantigen Interactions with T-cellAntigen Receptors.

The amino acid residues in contact with TCR are located in regions ofhigh sequence variability, presenting a unique surface for interactionwith the TCR. Residues implicated in TCR interactions by mutagenesis ofSEA and SEB reside in variable loop regions, while TSST-1 mutants thataffect TCR binding are mainly located in an α helix [Acharya, R. K. etal. (1994) Nature 367, 94-97; Kim, J. et al. (1994) Science 266,1870-1874]. Specifically, mutations that diminish T-cell receptorrecognition of SEB include residues N23, Y61, and the homologous SEA N25or Y64 (FIG. 2c). SEA residues S206 and N207 also control T-cellresponses [Hudson, et al.(1992) J. Exp. Med. 177: 175-184]. Mutants ofthe polar binding pocket, SEA Y92A and SEB Y89A, equivalently reducedT-cell responses (FIG. 2c), reflecting the observed decreases inDR1-binding (FIGS. 2a, b). While supporting reduced T-cell responses,mutants SEA Y64A and SEB Y61A retained normal affinities for DR1 (FIGS.2a-c).

EXAMPLE 5

Animal Models for Determining Biological Activity of BacterialSuperantigens: Mouse.

When compared to primates, mice are not very susceptible to the toxiceffects of SE, and we therefore sought to increase sensitivity with apotentiating dose of lipopolysaccharide (LPS) from Gram-negativebacteria [Stiles et al. (1993) Infect. Immun. 61, 5333-5338]. There wasno apparent effect in control animals injected with any of the SE (up to30 μg/mouse) or LPS (150 μg/mouse) alone (Table 1). Incrementalinjections of LPS were also not lethal, when given in amounts up to 250μg/mouse (data not shown). However, mice died between 24-48 h after SEand LPS were given to the same animal (Table 1). SEA was much more toxicthan either SEB or SEC1 and the calculated LD50 (μg toxin/kg) of SEA,SEB, and SEC1 with 95% fiducial limits was 18.5 (6.5, 38.5), 789.0(582.5, 1044.5), and 369.0 (197.5, 676.0), respectively.

TABLE 1 Titration of SEA, SEB, and SEC₁ in the C57BL/6 mouse lethalityassay % Lethality (no. of mice tested) with the following dose of SE, inmicrograms/mouse^(b): Stimulus^(a) 30 10 1 0.1  SEA + LPS 93 (15)^(b) 85(20) 80 (15) 20 (10)   SEB + LPS 80 (15) 27 (15)  0 (15) 0 (15) SEC₁ +LPS 80 (10) 60 (10) 10 (10) 0 (10) ^(a)LPS was injected into each mouse(150 ug) 4 h after the SE injection. Control mice injected with 150 ugof LPS (n = 20) or 30 ug of SEA, SEB, or SEC1 (n = 10) survived.^(b)Results are from a combination of separate experiments with fivemice per experiment.

The role of MHC class I and class II molecules in SE toxicity,potentiated by LPS, was addressed by using transgenic, MHC-deficientmice (Table 2). Class II-deficient animals were unaffected by a dose ofSE (30 μg) plus LPS (150 μg) that was lethal for 93% of wild-type and30% of class I-deficient mice. Mononuclear cells from class II-deficientanimals were not able to present SEA, as measured by proliferativeresponses. MHC class I-deficient cells were functional in supportingT-cell proliferation, but at levels <30% of the proliferative responsesupported by MHC-wild-type presenting cells (Table 3). Cell surfaceexpression levels were normal, when compared to nontransgenic C57BL/6,for A^(b) in class I-deficient mice, and K^(b)/D^(b) in classII-deficient mice. The T-cell responses of MHC class I- or classII-deficient mice were essentially equivalent to wild-type when SEA waspresented by mononuclear cells expressing both class I and II molecules(Table 3).

TABLE 2 Lethality of SEA and SEB in C57BL/6 mice lacking MHC class I orclass II % Lethality (no. of mice tested) with the following MHC classphenotype Stimulus^(a) I⁻II⁺ I⁺II⁻ I⁺II⁺ SEA + LPS 30 (10) 0 (5) 93 (15)SEA + LPS ND^(b) 0 (5) 80 (15) SEA only 0 (2) 0 (2) 0 (2) SEB onlyND^(b) 0 (2) 0 (2) LPS only 0 (5) 0 (5) 0 (5) ^(a)Mice were injectedwith 30 ug of SEA or SEB and, 4 h later, with 150 ug of LPS, asindicated. Control mice were injected with only SEA, SEB, or LPS.^(b)ND, not determined.

TABLE 3 Mouse T-cell responses to SEA are MHC class II-dependent T-cellresponses¹ T-cell/APC source³ 0.1 μg/ml SEA 1 μg/ml SEA Wild-typeC57/BL6 430,000 cpm² 700,000 cpm mouse/autologous MHC class I 117,000cpm 167,000 cpm knock-out C57/BL6 mouse/autologous MHC class II  8,000cpm  33,000 cpm knock-out C57/BL6 mouse/autologous Wild-type C57/BL6305,000 cpm 307,000 cpm mouse/wild-type MHC class I 420,000 cpm 445,000cpm knock-out C57/BL6 mouse/wild-type MHC class II 310,000 cpm 322,000cpm knock-out C57/BL6 mouse/wild-type ¹Cultures of mononuclear cellsderived from mouse spleens, cultured for 3 d with the indicated amountof SEA. ²Data represent the mean of triplicate determinations (<10 SEM)of [³H]thymidine incorporation. ³Antigen presenting cells (APC) wereisolated from spleens of the indicated mouse strain and added tocultures.

The serum levels of TNFα, IL-1α, IL-6, and IFNγ in mice injected withSEA, LPS, or SEA plus LPS were measured at various times followinginjection (FIG. 4). Compared to mice injected with either SEA or LPSalone, the serum levels of TNFα, IL-6, and IFNγ had increased 5-, 10-,and 15-fold, respectively, in animals given SEA plus LPS. SEA alone didnot elicit any detectable increase of TNFα, IL-6, or IFNγ abovebackground. In contrast to the other cytokines, IL-1α levels in miceinjected with SEA plus LPS resulted in a simple additive effect.

Serum levels of TNFα, IL-6, and IFNγ were maximal 2-4 h after the LPSinjection, but returned to normal by 10 h. The concentration of IL-1α inmice given SEA plus LPS had also peaked 2 h after the LPS injection, butstayed above background for the remaining determinations. Levels ofIL-1α in mice given only LPS or SEA peaked at 4 and 6 h, respectively.Unlike profiles for other cytokines, the highest amount of IL-1α in miceinjected with SEA and LPS corresponded to the peak stimulated by SEA,but not LPS.

This animal model was used in various stages of developing theinventions, as a means of assessing the physiological activity ofmutated superantigens. Control animals survived the maximum dose ofeither SE or LPS, while mice receiving both agents died. Wild-type SEAwas 43-fold more potent than SEB and 20-fold more potent than SEC1. Byusing BALB/c mice the toxicity of SEB was 10-20 fold higher. These dataconfirmed that the toxicity of SE was mainly exerted through a mechanismdependent on expression of MHC class II molecules and was linked tostimulated cytokine release. Thus this was a relevant preclinical modelthat could be used to predict human responses.

EXAMPLE 6

Animal Models for Determining Biological Activity of BacterialSuperantigens: Rhesus Monkey

The physiological responses of the rhesus monkey to bacterialsuperantigens is probably identical to humans, with the exception ofsensitivity [Bavari and Ulrich (1995) Clin. Immunol. Immunopath.76:248]. Generally SEB intoxicated monkeys developed gastrointestinalsigns within 24 hours post-exposure. Clinical signs were mastication,anorexia, emesis and diarrhea. Following mild, brief, self-limitinggastrointestinal signs, monkeys had a variable period of up to 40 hoursof clinical improvement. At approximately 48 hours post-exposure,intoxicated monkeys generally had an abrupt onset of rapidly progressivelethargy, dyspnea, and facial pallor. If given a lethal dose, deathoccurs within four hours of onset of symptoms. Only SEB has been used inchallenges of rhesus monkeys to determine physiological/pathologicaleffects. Human responses to bacterial superantigens are characterized bya rapid drop in blood pressure, elevated temperature, and multiple organfailure-the classical toxic shock syndrome (TSS). However, therespiratory route of exposure may involve some unique mechanisms. Theprofound hypotension characteristic of TSS is not observed, andrespiratory involvement is rapid, unlike TSS. Fever, prominent afteraerosol exposure, is generally not observed in cases of SEB ingestion.

EXAMPLE 7

Targeting Receptor Interactions to Develop Vaccines.

The SEA mutants Y92A, with reduced DR1 binding, and Y64A, with reducedTCR interactions, and K14E with wild-type (control) activity were usedto determine the correct receptor to target for vaccine development. Thebinding of WT or mutant SEA was evaluated with the MHC class IIexpressing murine B-cell lymphoma cell line A20 (Table 4). The bindingaffinity of WT SEA to mouse MHC class II (H-2d) molecules was lower thanthat observed with human MHC class II expressing cells, reflecting thereduced toxicity that bacterial SAgs exert in mice. WT SEA, Y64A andK14E all had the same relative affinity to mouse MHC class II molecules.Similar to the results obtained with human MHC class II molecules, theY92A mutant exhibited substantially reduced binding to A20 cells (Table4).

TABLE 4 Biological activity of superantigen vaccines MHC class II T-celltoxin T-cell anergy¹ binding² response SEA ++++ +++ +++ wild type TCRattenuated + +++ +/− Y64A MHC attenuated − +/− +/− Y92A Control ++++ ++++++ K14E ¹Based on attenuation of T-cell response to wild-type SEA inmice immunized with the mutant or wild-type SEA. ²Binding to the mouseMHC class II+ A20 cells, measured by flow cytometry

The effect of WT SEA or site-specific SEA mutants on splenic mononuclearcells obtained from nonimmunized C57BL/6 (H-2^(b)) mice is summarized inTable 4. Both WT SEA and the control mutant K14E were potent T cellactivators, effective at minimal concentrations of 10 to 100 pg/mL.However, T-cell responses to Y92A were reduced at least 100-fold,compared to SEA wild type, while Y64A-stimulated responses were slightlyhigher than Y92A. These results confirmed that attenuation ofsuperantigen binding to either MHC class II or TCR molecules resulted indramatically reduced mouse T-cell proliferation. These results mayindicate that the altered toxin may compete with wild type toxin for TCRbinding.

SEA WT (10 LD50), site-specific SEA mutants (10 μg/mouse each) or LPS(150 μg/mice) injected alone were nonlethal to mice (Table 5). However,combining LPS with either WT SEA or mutant K14E resulted in 100%lethality. For those mice receiving both LPS and WT or K14E SEA, 80%were dead by 24 h and 100% by 48 h. In contrast, 100% of Y92A and 80% ofY64A injected mice (coadministered with LPS) survived. The average timeto death for the 20% of mice that did not survive Y64A injectionoccurred at 48 to 72 h. These in vivo data correlated well with theresults obtained with the lymphocyte cultures. It was concluded that theobserved attenuation of toxicity in mice was a direct result of thereduced T-cell proliferation.

TABLE 5 Biologic effect of wild type (WT) staphylococcal enterotoxin A(SEA) and SEA mutants. Protein No. live/total WT 0/10 K14E 0/10 Y64A8/10 Y92A 10/10  NOTE. Mice were given 10 LD₅₀ (10 ug) of WT or mutantSEA. Lipopolysaccharide (150 ug/mouse) was injected 3 h later.

Having established that attenuation of receptor binding resulted inreduced toxicity, we next examined the immunogenicity of the SEAmutants. Mice were immunized with WT or mutant SEA. Control micereceived adjuvant only or were left untreated. One week before challengewith WT SEA, mice were bled and serum antibody titers were determinedfor each group (Table 6). Mice immunized with the 2 μg of Y64A or Y92Ahad serum antibody titers of 1:5000 and 1:1000, respectively.Immunization with 2 μg of WT SEA or control mutant resulted in titers of1:5,000 and 1:10,000, respectively. The highest immunizing dose (10μg/mouse) was most effective for all animals, resulting in antibodytiters which were greater than 1:10,000. All mice were challenged with10 LD50 of WT SEA (potentiated with LPS). The survival data correlatedwell with the levels of serum antibodies in immunized mice. All micethat were vaccinated with 10 μg of Y64A or Y92A, survived the lethalchallenge dose of WT SEA. Slightly less protection was afforded by thelower vaccination dose of mutant Y64A or Y92A. All mice immunized withboth doses of WT SEA survived the lethal challenge with WT potentiatedwith LPS. Mice immunized with mutant K14E exhibited survivals of 100%and 80% for high and low vaccination doses, respectively. Allnonimmunized or control mice that were vaccinated with adjuvant alonedied when challenged with WT SEA and a potentiating dose of LPS.

TABLE 6 Mice immunized with attenuated forms of staphylococcalenterotoxin A (SEA) produce high titers of neutralizing antibody.Immunizing Dose Anti-SEA agent (ug/mouse) antibody titer* No. live/totalWT 2 10,000-50,000 10/10 10 10,000-50,000 10/10 K14E 2  5,000-10,000 8/10 10 10,000-50,000 10/10 Y64A 2  5,000-10,000  6/10 10 10,000-50,00010/10 Y92A 2 1,000-5,000  2/10 10 10,000-50,000 10/10 Adjuvant  50-100 0/10 NOTE. Mice were given 10 LD₅₀ of wild type (WT) SEA challengefollowed by potentiating dose of lipopolysaccharide (150 ug/mouse) 3 hlater. *Reciprocal of serum dilution resulting in optical densityreading four times above negative controls (wells containing either noSEA or no primary antibody).

EXAMPLE 8

Immune Recognition of SAg Mutants.

Bacterial SAgs induce clonal anergy of specific subsets of T cells inmice. It was possible that the loss of sensitivity to WT SEA among themice vaccinated with the attenuated mutant forms represented a state ofspecific non-responsiveness instead of specific immunity. To addressthis issue, lymphocyte responses to SEA WT were measured with splenicmononuclear cells collected 2 weeks after the third immunization. Asexpected, lymphocytes from mice that were immunized with WT SEA orcontrol SEA mutant showed little to no proliferation when incubated withthe WT SAg. In contrast, lymphocytes obtained from control mice or thoseimmunized with either Y64A or Y92A all responded vigorously to the WTSEA (FIG. 5). The TCRs used by T cells from the SEA-vaccinated mice werethen characterized by flow cytometry. T cells from immunized or controlmice were incubated with WT SEA in culture for 7 days, followed by a 5day expansion in IL-2 containing medium. Distinct populations ofactivated TCR Vβ11 positive cells were observed with T cells from miceimmunized with Y92A and Y64A, representing 48% and 40% of T cells,respectively. However, Vβ11 expressing cells obtained from SEA WT orK14E immunized mice were about 1% and 6% of the total T-cell population,respectively, suggesting that this subset was nonresponsive torestimulation with the WT SAg. T cells bearing Vβ 17a, 3, 7, and 10bwere unchanged for all mice. It was apparent that T-cell responses toboth the TCR and MHC class II binding-attenuated SEA mutants weresimilar to each other, but differed from responses to control or WTmolecules. These results suggested that an alternative, perhapsconventional antigen processing mechanism was functioning inpresentation of the SAg mutants Y64A and Y92A.

EXAMPLE 9

Rhesus Monkey Immunizations with Monovalent Vaccines.

The SEA vaccine L48R, Y89A, D70R (A489270)(SEQ ID NO:12) and SEB vaccineY89A, Y94A, L45R (B899445)(SEQ ID NO:8) were used to immunize rhesusmonkeys. The animals received a total of three i.m. injections (10-20μg/animal), given at monthly intervals. Rhesus monkeys that wereinjected with these vaccines had no detectable increase of serumcytokines and no apparent toxicity. The serological response of animalsvaccinated with three doses of formalin-treated SEB toxoid (100μg/injection) gave results comparable to one or two injections withB899445 (Table 7), suggesting that the recombinant vaccines were veryimmunogenic. Immunized rhesus monkeys survived a lethal challengewith >10 LD50 of wild-type SEB (Table 7, 8). Collectively, these resultssuggest that the engineered SEB vaccine is safe, highly antigenic andeffective at protecting the immunized individual from lethal aerosolexposure to SEB.

TABLE 7 Rhesus monkey antibody responses to vaccine B899445; Oneinjection of B899445 outperforms three injections of SEB toxoid SurvivalSEB Antibody % Inhibition of >20 × LD50 Vaccine¹/animal # response²T-cell response³ challenge⁴ preimmune sera/ 0.161 5 dead pooled toxoid/10.839 0 dead toxoid/2 0.893 34 live toxoid/3 1.308 57 live toxoid/41.447 55 live B899445/1 1.788 69 live B899445/2 0.78 49 live ¹Rhesusmonkeys were immunized with one dose (20 μg injection) of B899445vaccine or three doses of formalin-treated SEB toxoid (100 μg/injection)one month apart; both used Alum adjuvants. ²Sera were collected onemonth after the final injection. Antibody responses were determined byELISA and the results are shown as mean optical densities of triplicatewells (±SEM). ³Rhesus monkey T. cells, obtained from an untreatedanimal, were preincubated with diluted (1:70) serum from immunizedmonkeys and then cultured with wild type SEB. Data are shown as % of Tcell responses, where serum of rhesus monkey injected with adjuvant onlyrepresented the 100% of response to wild type SEB. ⁴Rhesus monkeys werechallenged by aerosol exposure and monitered for four days.

TABLE 8 Engineered staphylococcal entero- toxin B vaccine efficacy inrhesus monkeys Treatment¹ Antibody titer² Immune protection³Vaccine >10,000 100% with adjuvant Adjuvant only <50 0% ¹Rhesus monkeys(n = 10) were injected i.m. with 10 μg of SEB vaccine with Alhydrogeladjuvant. A total of 3 immunizations, 1 month apart were given. Controls(n = 2) received only Alhydrogel. ²Serum dilution resulting in opticaldensity readings of four times above the negative control, consisting ofno SEB or serum added to the wells. ³Immunized and control rhesusmonkeys were challenged with >10 LD50 of wild-type staphylococcalenterotoxin B as an aerosol.

Serum from monkeys that were immunized with the genetically attenuatedvaccine inhibited T-lymphocyte responses to wild type SEB (Table 7)similarly or better than monkeys that received the SEB toxoid.Collectively, these results suggest that the recombinant SAg vaccinesare safe, highly antigenic, and induce protective immunity.

Serum from B899445 immunized rhesus monkeys blocked human lymphocyteresponses to wild-type superantigen when tested in ex vivo cultures(Table 7). These data again showed that the second and third injectionsof vaccine were approximately equivalent in stimulating neutralizingantibody responses. Normal T-cell responses to several superantigens,including the wild-type protein, were observed in immunized animals,indicating that no specific or generalized anergy occurred (FIG. 6).

EXAMPLE 10

A. Multivalent Superantigen Vaccines: Rhesus Monkey Immunizations.

Rhesus monkeys were immunized with a combined vaccine consisting ofB899445 and A489270. Following the third injection, antibody recognitionof wild-type bacterial superantigens was examined (FIG. 7). High titersof anti-SEB, SEC1 and SEA antibodies were evident.

B. Mouse Immunizations.

Mice (BALB/c) were immunized with a combined vaccine consisting of SEA,SEB, SEC1 and TSST-1 (all wild-type). The antibody responses againsteach individual superantigen were assessed (Table 9). Antibodies wereinduced against each of the component antigens, providing sufficientlevels to protect the mice from a lethal challenge of superantigen,potentiated with LPS. Although not shown in the Table, antibodyresponses against SPE-A were also observed. Mice were also immunizedwith individual superantigens and antibody responses against othersuperantigens were measured (Table 10). Each individual immunogeninduced partial or complete protective antibody responses against allother superantigens tested.

TABLE 9 Superantigen cross-reactivity of antibodies from mice immunizedwith individual bacterial superantigens Immunizing¹ Challenging² ELISA³Neutralizing⁴ Toxin Toxin Titer Antibody SEA SEA >1/25,000 100% SEASEB >1/25,000 100% SEA SEC1 >1/25,000 100% SEA TSST1 >1/10,000 100% SEBSEB >1/25,000 100% SEB SEA >1/10,000 100% SEB SEC1 >1/2,500 100% SEBTSST1 >1/10,000 100% SEC1 SEC1 >1/10,000 100% SEC1 SEA >1/10,000 100%SEC1 SEB >1/25,000 100% SEC1 TSST1 >1/10,000 100% TSST1 TSST1 <1/10,000100% TSST1 SEA <1/1,000 50% TSST1 SEB <1/1,000 40% TSST1 SEC1 <1/1,00040% ¹Three injections with 20 ug of antigen (BALB/c mice).²LPS-potentiated challenge with 10 LD₅₀s of superantigen. ³ELISAantibody response against an individual superantigen. ⁴Percent micesurviving an LPS-potentiated challenge (n = 10).

TABLE 10 Multivalent superantigen vaccine. Mouse immune responses.Immunizing Challenging Antibody toxin¹ toxin² Titer³ % survival SE-A, B,C1, TSST-1 all N/A 100% ″ ″ SEA >25,000 100% ″ ″ SEB >25,000 100% ″ ″SEC1 >25,000 100% ″ ″ TSST-1  >6,400 100% ¹Total of three injections,two weeks apart, in RIBI adjuvant. ²>10 × LD50, potentiated with E. coillipopolysaccharide. ³Measured by ELISA.

EXAMPLE 11

Design of Altered TSST-1 Toxin Vaccine, TST30.

A comprehensive study of the relationships of TSST-1 protein structureto receptor binding were undertaken to provide insight into the designof the vaccine TST30. We have discovered that TSST-1 interactions withthe human MHC class II receptor, HLA-DR, are relatively weak and can bedisrupted by altering only a single critical amino acid residue of thetoxin. Site-directed mutagenesis of a gene encoding the toxin andexpression of the new protein product in E. coli were then used to testthe design of the vaccine. The TSST-1 gene used was contained within afragment of DNA isolated by BglI restriction enzyme digestion of thegene isolated from a toxigenic strain of Staphylococcus aureus (AB259;Kreiswirth and Novick (1987) Mol. Gen. Genet. 208, 84-87). The sequenceof this gene is identical to all currently known TSST-1 isolates ofhuman origin. The wild-type TSST-1 gene can be readily cloned from anumber of clinical S. aureus isolates. The DNA fragment containing theTSST-1 gene was isolated by agarose gel electrophoresis and ligated inotthe prokaryotic expression vector pSE380 (Invitrogen Corp.). The DNAclone consisted of sequences encoding the leader peptide and the fulllength of the mature TSST-1 protein(SEQ ID NO:11). This engineeredvaccine is currently being evaluated to determine mouse and human T-cellreactivities in vitro, and lethality in mice. The TST30 vaccine consistsof the following mutation introduced into the toxin molecule: leucine atamino acid residue 30 changed to arginine. Two other mutations, namelyAsp27 to Ala and Ile46 to Ala have also been designed. The final vaccinemay incorporate one or both of these additional mutations.

The binding interface between TSST-1 and HLA-DR consists of a largerelatively flat surface located in the N-terminal domain. Leucine 30protrudes from a reverse turn on the surface of TSST-1 and forms themajor hydrophobic contact with the HLA-DR receptor molecule. Mutation ofthe single residue leucine 30 in TSST-1 to the charged amino acid sidechain of arginine is predicted to disrupt this major contact with thereceptor molecule, resulting in a significant reduction in DR1 binding.This mutant molecule should therefore have lost the toxin attributes ofthe wild-type molecule.

TST30 (SEQ ID NO:12) was expressed as a recombinant protein in E.coli,as either a periplasmically secreted protein or as a cytoplasmicproduct. Purification was achieved by immunoaffinity chromatography orpreparative isoelectric focusing after an initial ion-exchangeCM-Sepharose enrichment step. The method of purification was notcritical to the performance of the vaccine. Lipopolysaccharidecontaminants, resulting from expression in a Gram-negative bacterium,were readily removed (as determined by limulus assay) using a variety ofstandard methods. The final purified vaccine is not toxic to mice atlevels equivalent to 10 LD₅₀ of the native TSST-1. No indicators oftoxicity were found in surrogate assays of human T-cell stimulation.

Conclusive vaccine studies demonstrating that TST30 is highly antigenicand induces protective immunity are in progress in a mouse animal model.Mouse lethality is achieved at less than 1 ug/animal when a potentiatingsignal like lipopolysaccharide from Gram-negative bacteria (LPS) isprovided. When coadministered with LPS, wild-type TSST-1 is 100% lethalto mice (10 LD₅₀). Mice receive three injections (two weeks betweeninjections) of 20 ug/mouse in alhydrogel and protection against thelethal effects of 10 LD₅₀ of TSST-1 are assessed.

EXAMPLE 12

Design of Altered SPEA Toxin Vaccine, SPEa42

The SPEa interactions with human MHC class II receptor, HLA-DR, arerelatively weak and can be disrupted by altering only a single criticalamino acid residue of the toxin. Site-directed mutagenesis of a geneencoding the toxin and expression of the new protein product in E.coliwere then used to test the design of the vaccine. The SPEa gene used wasclone from a SPEa-toxigenic strain of Streptococcus by using specificDNA oligonucleotide primers and the polymerase chain reaction method.The sequence of this gene is identical to SPEa isolates of human originknown within the public domain. The DNA fragment containing the SPEagene was isolated by agarose gel electrophoresis and ligated into aprokaryotic expression vector (pETx or pSE380). The DNA clone consistedof sequences encoding the leader peptide and the full length of themature SPEa protein or SPEa42 (SEQ ID NO:15) without a leader sequence.We recognize that there are additional ways to express or produce themature SPEa vaccine. The SPEa vaccine consists of the following mutationintroduced into the toxin molecule: leucine at amino acid residue 42changed to arginine.

The binding interface between SPEa and HLA-DR is predicted to consist ofcontacts located in the N-terminal domain that are conserved with otherbacterial superantigens. Leucine 42 of SPEa is predicted to protrudefrom a reverse turn on the surface of SPEa and form a major hydrophobiccontact with the HLA-DR receptor molecule. Mutation of the singleresidue leucine 42 in SPEa to the charged amino acid side chain ofarginine is predicted to disrupt this major contact with the receptormolecule, resulting in a significant reduction in DR1 binding. Thismutant molecule should therefore have lost the toxin attributes of thewild-type molecule.

SPEa42 (SEQ ID NO:16) was expressed as a recombinant protein in E.coli,as either a periplasmically secreted protein or as a cytoplasmicproduct. Purification was achieved by immunoaffinity chromatography orpreparative isoelectric focusing after an initial ion-exchangeCM-Sepharose enrichment step. The method of purification was notcritical to the performance of the vaccine. Lipopolysaccharidecontaminants, resulting from expression in a Gram-negative bacterium,were readily removed (as determined by limulus assay) using a variety ofstandard methods. The final purified vaccine is not toxic to mice atlevels equivalent to 10 LD₅₀ of the native TSST-1. No indicators oftoxicity were found in surrogate assays of human T-cell stimulation.

Conclusive vaccine studies demonstrating that SPEa42 is highly antigenicand induces protective immunity are in progress in a mouse animal model.Mouse lethality is achieved at less than 1 ug/animal when a potentiatingsignal like lipopolysaccharide from Gram-negative bacteria (LPS) isprovided. When coadministered with LPS, wild-type SPEa is 100% lethal tomice (10 LD₅₀). Mice receive three injections (two weeks betweeninjections) of 20 ug/mouse in alhydrogel and protection against thelethal effects of 10 LD₅₀ of SPEa are assessed

EXAMPLE 13

Design of Altered Superantigen Toxin Vaccine, SEC45

For Staphylococcal enterotoxin C1 (SEC1), the leucine at position 45 waschanged to lysine (SEC45)(SEQ ID NO:14). This mutation is anticipated toprevent SEC1 from interacting with the MHC class II receptor bysterically blocking the hydrophobic loop (centered around leucine 45)from binding to the alpha chain of the receptor. SEC1 is more closelyhomologous to SEB than SEA or the other superantigen toxins. Thepresence of zinc in SEC1 may impart additional binding characteristicsthat allow, in some cases, this superantigen toxin to bind to T-cellantigen receptors without the required MHC class II moleculeinteractions. To circumvent the binding to T-cell antigen receptors,mutations of SEC1 residues N23 (changed to alanine), V91 (changed tolysine) are being performed.

                   #             SEQUENCE LISTING #   x(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES:25(2) INFORMATION FOR SEQ ID NO:1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 830           (B) TYPE: Nucleic Acid          (C) STRANDEDNESS: Unknown           (D) TOPOLOGY: Unknown    (ii) MOLECULE TYPE: DNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:atgaaaaaaa cagcatttac attactttta ttcattgccc      #                  #    40 taacgttgac aacaagtcca cttgtaaatg gtagcgagaa     #                   #    80aagcgaagaa ataaatgaaa aagatttgcg aaaaaagtct      #                  #   120 gaattgcagg gaacagcttt aggcaatctt aaacaaatct     #                   #   160attattacaa tgaaaaagct aaaactgaaa ataaagagag      #                  #   200 tcacgatcaa tttcgacagc atactatatt gtttaaaggc     #                   #   240ttttttacag atcattcgtg gtataacgat ttattagtac      #                  #   280 gttttgattc aaaggatatt gttgataaat ataaagggaa     #                   #   320aaaagtagac ttgtatggtg cttatgctgg ttatcaatgt      #                  #   360 gcgggtggta caccaaacaa aacagcttgt atgtatggtg     #                   #   400gtgtaacgtt acatgataat aatcgattga ccgaagagaa      #                  #   440 aaaagtgccg atcaatttat ggctagacgg taaacaaaat     #                   #   480acagtacctt tggaaacggt taaaacgaat aagaaaaatg      #                  #   520 taactgttca ggagttggat cttcaagcaa gacgttattt     #                   #   560acaggaaaaa tataatttat ataactctga tgtttttgat      #                  #   600 gggaaggttc agaggggatt aatcgtgttt catacttcta     #                   #   640cagaaccttc ggttaattac gatttatttg gtgctcaagg      #                  #   680 acagtattca aatacactat taagaatata tagagataat     #                   #   720aaaacgatta actctgaaaa catgcatatt gatatatatt      #                  #   760 tatatacaag ttaaACATGG TAGTTTTGAC CAACGTAATG     #                   #   800 TTCAGATTAT TATGAACCGA GAATAATCTA         #                   #          830 (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 257          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:Met Lys Lys Thr Ala Phe Thr Leu Leu Leu 1                5  #                 10 Phe Ile Ala Leu Thr Leu Thr Thr Ser Pro                15   #                 20Leu Val Asn Gly Ser Glu Lys Ser Glu Glu                 25  #                 30 Ile Asn Glu Lys Asp Leu Arg Lys Lys Ser                35   #                 40Glu Leu Gln Gly Thr Ala Leu Gly Asn Leu                 45  #                 50 Lys Gln Ile Tyr Tyr Tyr Asn Glu Lys Ala                55   #                 60Lys Thr Glu Asn Lys Glu Ser His Asp Gln                 65  #                 70 Phe Arg Gln His Thr Ile Leu Phe Lys Gly                75   #                 80Phe Phe Thr Asp His Ser Trp Tyr Asn Asp                 85  #                 90 Leu Leu Val Arg Phe Asp Ser Lys Asp Ile                95   #                100Val Asp Lys Tyr Lys Gly Lys Lys Val Asp                 105  #               110 Leu Tyr Gly Ala Tyr Ala Gly Tyr Gln Cys                115   #               120Ala Gly Gly Thr Pro Asn Lys Thr Ala Cys                 125  #               130 Met Tyr Gly Gly Val Thr Leu His Asp Asn                135   #               140Asn Arg Leu Thr Glu Glu Lys Lys Val Pro                 145  #               150 Ile Asn Leu Trp Leu Asp Gly Lys Gln Asn                155   #               160Thr Val Pro Leu Glu Thr Val Lys Thr Asn                 165  #               170 Lys Lys Asn Val Thr Val Gln Glu Leu Asp                175   #               180Leu Gln Ala Arg Arg Tyr Leu Gln Glu Lys                 185  #               190 Tyr Asn Leu Tyr Asn Ser Asp Val Phe Asp                195   #               200Gly Lys Val Gln Arg Gly Leu Ile Val Phe                 205  #               210 His Thr Ser Thr Glu Pro Ser Val Asn Tyr                215   #               220Asp Leu Phe Gly Ala Gln Gly Gln Tyr Ser                 225  #               230 Asn Thr Leu leu Arg Ile Tyr Arg Asp Asn                235   #               240Lys Thr Ile Asn Ser Glu Asn Met His Ile                 245  #               250 Asp Ile Tyr Leu Tyr Thr Ser                 255(2) INFORMATION FOR SEQ ID NO:3:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 757           (B) TYPE: Nucleic Acid          (C) STRANDEDNESS: Unknown           (D) TOPOLOGY: Unknown    (ii) MOLECULE TYPE: DNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:atgagaaaag cgaagaaata aatgaaaaag atttgcgaaa      #                  #    40 aaagtctgaa ttgcagggaa cagctttagg caatcttaaa     #                   #    80Caaatctatt attacaatga aaaagctaaa actgaaaata      #                  #   120 aagagagtca cgatcaattt cgacagcata ctatattgtt     #                   #   160taaaggcttt tttacagatc attcgtggta taacgattta      #                  #   200 ttagtacgtt ttgattcaaa ggatattgtt gataaatata     #                   #   240aagggaaaaa agtagacttg tatggtgctt atgctggtta      #                  #   280 tcaatgtgcg ggtggtacac caaacaaaac agcttgtatg     #                   #   320tatggtggtg taacgttaca tgataataat cgattgaccg      #                  #   360 aagagaaaaa agtgccgatc aatttatggc tagacggtaa     #                   #   400acaaaataca gtacctttgg aaacggttaa aacgaataag      #                  #   440 aaaaatgtaa ctgttcagga gttggatctt caagcaagac     #                   #   480gttatttaca ggaaaaatat aatttatata actctgatgt      #                  #   520 ttttgatggg aaggttcaga ggggattaat cgtgtttcat     #                   #   560acttctacag aaccttcggt taattacgat ttatttggtg      #                  #   600 ctcaaggaca gtattcaaat acactattaa gaatatatag     #                   #   640agataataaa acgattaact ctgaaaacat gcatattgat      #                  #   680 atatatttat atacaagtta aACATGGTAG TTTTGACCAA     #                   #   720CGTAATGTTC AGATTATTAT GAACCGAGAA TAATCTA       #                  #     757 (2) INFORMATION FOR SEQ ID NO:4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH:233          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:Met Glu Lys Ser Glu Glu Ile Asn Glu Lys                  5 #                   #10 Asp Leu Arg Lys Lys Ser Glu Leu Gln Gly                15   #                 20Thr Ala Leu Gly Asn Leu Lys Gln Ile Tyr                 25  #                 30 Tyr Tyr Asn Glu Lys Ala Lys Thr Glu Asn                35   #                 40Lys Glu Ser His Asp Gln Phe Arg Gln His                 45  #                 50 Thr Ile Leu Phe Lys Gly Phe Phe Thr Asp                55   #                 60His Ser Trp Tyr Asn Asp Leu Leu Val Arg                 65  #                 70 Phe Asp Ser Lys Asp Ile Val Asp Lys Tyr                75   #                 80Lys Gly Lys Lys Val Asp Leu Tyr Gly Ala                 85  #                 90 Tyr Ala Gly Tyr Gln Cys Ala Gly Gly Thr                95   #                100Pro Asn Lys Thr Ala Cys Met Tyr Gly Gly                 105  #               110 Val Thr Leu His Asp Asn Asn Arg Leu Thr                115   #               120Glu Glu Lys Lys Val Pro Ile Asn Leu Trp                 125  #               130 Leu Asp Gly Lys Gln Asn Thr Val Pro Leu                135   #               140Glu Thr Val Lys Thr Asn Lys Lys Asn Val                 145  #               150 Thr Val Gln Glu Leu Asp Leu Gln Ala Arg                155   #               160Arg Tyr Leu Gln Glu Lys Tyr Asn Leu Tyr                 165  #               170 Asn Ser Asp Val Phe Asp Gly Lys Val Gln                175   #               180Arg Gly Leu Ile Val Phe His Thr Ser Thr                 185  #               190 Glu Pro Ser Val Asn Tyr Asp Leu Phe Gly                195   #               200Ala Gln Gly Gln Tyr Ser Asn Thr Leu Leu                 205  #               210 Arg Ile Tyr Arg Asp Asn Lys Thr Ile Asn                215   #               220Ser Glu Asn Met His Ile Asp Ile Tyr Leu                 225  #               230 Tyr Thr Ser (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1712          (B) TYPE: Nucleic Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:GAACTAGGTA GAAAAATAAT TATGAGAAAA CACTATGTTG      #                  #    40 TTAAAGATGT TTTCGTATAT AAGTTTAGGT GATGTATAGT     #                   #    80TACTTAATTT TAAAAGCATA ACTTAATTAA TATAAATAAC      #                  #   120 ATGAGATTAT TAAATATAAT TAAGTTTCTT TTAATGTTTT     #                   #   160TTTAATTGAA TATTTAAGAT TATAACATAT ATTTAAAGTG      #                  #   200 TATCTAGATA CTTTTTGGGA ATGTTGGATA AAGGAGATAA     #                   #   240AAAATGTATA AGAGATTATT TATTTCACAT GTAATTTTGA      #                  #   280 TATTCGCACT GATATTAGTT ATTTCTACAC CCAACGTTTT     #                   #   320AGCAGAGAGT CAACCAGATC CTAAACCAGA TGAGTTGCAC      #                  #   360 AAATCGAGTA AATTCACTGG TTTGATGGAA GATATGAAAG     #                   #   400TTTTGTATGA TGATAATCAT GTATCAGCAA TAAACGTTAA      #                  #   440 ATCTATAGAT CAATTTCTAT ACTTTGACTT AATATATTCT     #                   #   480ATTAAGGACA CTAAGTTAGG GGATTATGAT AATGTTCGAG      #                  #   520 TCGAATTTAA AAACAAAGAT TTAGCTGATA AATACAAAGA     #                   #   560TAAATACGTA GATGTGTTTG GAGCTAATTA TTATTATCAA      #                  #   600 TGTTATTTTT CTAAAAAAAC GAATGATATT AATTCGCATC     #                   #   640AAACTGACAA ACGAAAAACT TGTATGTATG GTGGTGTAAC      #                  #   680 TGAGCATAAT GGAAACCAAT TAGATAAATA TAGAAGTATT     #                   #   720ACTGTTCGGG TATTTGAAGA TGGTAAAAAT TTATTATCTT      #                  #   760 TTGACGTACA AACTAATAAG AAAAAGGTGA CTGCTCAAGA     #                   #   800ATTAGATTAC CTAACTCGTC ACTATTTGGT GAAAAATAAA      #                  #   840 AAACTCTATG AATTTAACAA CTCGCCTTAT GAAACGGGAT     #                   #   880ATATTAAATT TATAGAAAAT GAGAATAGCT TTTGGTATGA      #                  #   920 CATGATGCCT GCACCAGGAG ATAAATTTGC CCAATCTAAA     #                   #   960TATTTAATGA TGTACAATGA CAATAAAATG GTTGATTCTA      #                  #  1000 AAGATGTGAA GATTGAAGTT TATCTTACGA CAAAGAAAAA     #                   #  1040GTGAAATTAT ATTTTAGAAA AGTAAATATG AAGAGTTAGT      #                  #  1080 AATTAAGGCA GGCACTTATA GAGTACCTGC CTTTTCTAAT     #                   #  1120ATTATTTAGT TATAGTTATT TTTGTTATAT CTCTCTGATT      #                  #  1160 TAGCATTAAC CCCTTGTTGC CATTATAGTT TTCACCAACT     #                   #  1200TTAGCTGAAA TTGGGGGATC ATTTTTATCT TTACTATGGA      #                  #  1240 TAGTTACTGT GTCGCCGTTT TTAACGATTT GTTTCTCTTT     #                   #  1280TAATTTGTCA GTTAATTTTT TCCATGCATC ATTTGCGTCA      #                  #  1320 AACCTATTTC CATTTGGATT TATTCTTGAC AAATCAATTC     #                   #  1360TTTTAACACT ATCGGTATTA ATCGGCTTGT TATTAAAATT      #                  #  1400 ACTAAGTTCA TCTAAATCAG CTGTACCCGT AATACTACTT     #                   #  1440TCGCCACCAT TATTTAAATT GTACGTAACA CCAACTGTCT      #                  #  1480 CATTTGCTGT TTTATCGATA ATATTTGCTT CTTTCAAAGC     #                   #  1520ATCTCTTACA TTTTTCCATA AGTCTCTATC TGTTATTTCA      #                  #  1560 GAAGCCTTTG CAACGTTATT AATACCATTA TAATTTGAAG     #                   #  1600AAGAATGAAA ACCTGAACCT ACTGTTGTTA AAACTAAAGC      #                  #  1640 ACTTGCTATC AATGTTCTTG TTAATAGTTT TTTATTCATT     #                   #  1680 TTATTTTCTC CTATAACTTA TTTGCAATCG AT       #                   #        1712 (2) INFORMATION FOR SEQ ID NO:6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 266          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:Met Tyr Lys Arg Leu Phe Ile Ser His Val                  5 #                   #10 Ile Leu Ile Phe Ala Leu Ile Leu Val Ile                15   #                 20Ser Thr Pro Asn Val Leu Ala Glu Ser Gln                 25  #                 30 Pro Asp Pro Lys Pro Asp Glu Leu His Lys                35   #                 40Ser Ser Lys Phe Thr Gly Leu Met Glu Asp                 45  #                 50 Met Lys Val Leu Tyr Asp Asp Asn His Val                55   #                 60Ser Ala Ile Asn Val Lys Ser Ile Asp Gln                 65  #                 70 Phe Leu Tyr Phe Asp Leu Ile Tyr Ser Ile                75   #                 80Lys Asp Thr Lys Leu Gly Asp Tyr Asp Asn                 85  #                 90 Val Arg Val Glu Phe Lys Asn Lys Asp Leu                95   #                100Ala Asp Lys Tyr Lys Asp Lys Tyr Val Asp                 105  #               110 Val Phe Gly Ala Asn Tyr Tyr Tyr Gln Cys                115   #               120Tyr Phe Ser Lys Lys Thr Asn Asp Ile Asn                 125  #               130 Ser His Gln Thr Asp Lys Arg Lys Thr Cys                135   #               140Met Tyr Gly Gly Val Thr Glu His Asn Gly                 145  #               150 Asn Gln Leu Asp Lys Tyr Arg Ser Ile Thr                155   #               160Val Arg Val Phe Glu Asp Gly Lys Asn Leu                 165  #               170 Leu Ser Phe Asp Val Gln Thr Asn Lys Lys                175   #               180Lys Val Thr Ala Gln Glu Leu Asp Tyr Leu                 185  #               190 Thr Arg His Tyr Leu Val Lys Asn Lys Lys                195   #               200Leu Tyr Glu Phe Asn Asn Ser Pro Tyr Glu                 205  #               210 Thr Gly Tyr Ile Lys Phe Ile Glu Asn Glu                215   #               220Asn Ser Phe Trp Tyr Asp Met Met Pro Ala                 225  #               230 Pro Gly Asp Lys Phe Ala Gln Ser Lys Tyr                235   #               240Leu Met Met Tyr Asn Asp Asn Lys Met Val                 245  #               250 Asp Ser Lys Asp Val Lys Ile Glu Val Tyr                255   #               260 Leu Thr Thr Lys Lys Lys                265 (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1712          (B) TYPE: Nucleic Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:GAACTAGGTA GAAAAATAAT TATGAGAAAA CACTATGTTG      #                  #    40 TTAAAGATGT TTTCGTATAT AAGTTTAGGT GATGTATAGT     #                   #    80TACTTAATTT TAAAAGCATA ACTTAATTAA TATAAATAAC      #                  #   120 ATGAGATTAT TAAATATAAT TAAGTTTCTT TTAATGTTTT     #                   #   160TTTAATTGAA TATTTAAGAT TATAACATAT ATTTAAAGTG      #                  #   200 TATCTAGATA CTTTTTGGGA ATGTTGGATA AAGGAGATAA     #                   #   240AAAATGTATA AGAGATTATT TATTTCACAT GTAATTTTGA      #                  #   280 TATTCGCACT GATATTAGTT ATTTCTACAC CCAACGTTTT     #                   #   320AGCAGAGAGT CAACCAGATC CTAAACCAGA TGAGTTGCAC      #                  #   360 AAATCGAGTA AATTCACTGG TTTGATGGAA AATATGAAAG     #                   #   400TTTTGTATGA TGATAATCAT GTATCAGCAA TAAACGTTAA      #                  #   440 ATCTATAGAT CAATTTCGAT ACTTTGACTT AATATATTCT     #                   #   480ATTAAGGACA CTAAGTTAGG GAATTATGAT AATGTTCGAG      #                  #   520 TCGAATTTAA AAACAAAGAT TTAGCTGATA AATACAAAGA     #                   #   560TAAATACGTA GATGTGTTTG GAGCTAATGC TTATTATCAA      #                  #   600 TGTGCTTTTT CTAAAAAAAC GAATGATATT AATTCGCATC     #                   #   640AAACTGACAA ACGAAAAACT TGTATGTATG GTGGTGTAAC      #                  #   680 TGAGCATAAT GGAAACCAAT TAGATAAATA TAGAAGTATT     #                   #   720ACTGTTCGGG TATTTGAAGA TGGTAAAAAT TTATTATCTT      #                  #   760 TTGACGTACA AACTAATAAG AAAAAGGTGA CTGCTCAAGA     #                   #   800ATTAGATTAC CTAACTCGTC ACTATTTGGT GAAAAATAAA      #                  #   840 AAACTCTATG AATTTAACAA CTCGCCTTAT GAAACGGGAT     #                   #   880ATATTAAATT TATAGAAAAT GAGAATAGCT TTTGGTATGA      #                  #   920 CATGATGCCT GCACCAGGAG ATAAATTTGA CCAATCTAAA     #                   #   960TATTTAATGA TGTACAATGA CAATAAAATG GTTGATTCTA      #                  #  1000 AAGATGTGAA GATTGAAGTT TATCTTACGA CAAAGAAAAA     #                   #  1040GTGAAATTAT ATTTTAGAAA AGTAAATATG AAGAGTTAGT      #                  #  1080 AATTAAGGCA GGCACTTATA GAGTACCTGC CTTTTCTAAT     #                   #  1120ATTATTTAGT TATAGTTATT TTTGTTATAT CTCTCTGATT      #                  #  1160 TAGCATTAAC CCCTTGTTGC CATTATAGTT TTCACCAACT     #                   #  1200TTAGCTGAAA TTGGGGGATC ATTTTTATCT TTACTATGGA      #                  #  1240 TAGTTACTGT GTCGCCGTTT TTAACGATTT GTTTCTCTTT     #                   #  1280TAATTTGTCA GTTAATTTTT TCCATGCATC ATTTGCGTCA      #                  #  1320 AACCTATTTC CATTTGGATT TATTCTTGAC AAATCAATTC     #                   #  1360TTTTAACACT ATCGGTATTA ATCGGCTTGT TATTAAAATT      #                  #  1400 ACTAAGTTCA TCTAAATCAG CTGTACCCGT AATACTACTT     #                   #  1440TCGCCACCAT TATTTAAATT GTACGTAACA CCAACTGTCT      #                  #  1480 CATTTGCTGT TTTATCGATA ATATTTGCTT CTTTCAAAGC     #                   #  1520ATCTCTTACA TTTTTCCATA AGTCTCTATC TGTTATTTCA      #                  #  1560 GAAGCCTTTG CAACGTTATT AATACCATTA TAATTTGAAG     #                   #  1600AAGAATGAAA ACCTGAACCT ACTGTTGTTA AAACTAAAGC      #                  #  1640 ACTTGCTATC AATGTTCTTG TTAATAGTTT TTTATTCATT     #                   #  1680 TTATTTTCTC CTATAACTTA TTTGCAATCG AT       #                   #        1712 (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 266          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:Met Tyr Lys Arg Leu Phe Ile Ser His Val                  5 #                   #10 Ile Leu Ile Phe Ala Leu Ile Leu Val Ile                15   #                 20Ser Thr Pro Asn Val Leu Ala Glu Ser Gln                 25  #                 30 Pro Asp Pro Lys Pro Asp Glu Leu His Lys                35   #                 40Ser Ser Lys Phe Thr Gly Leu Met Glu Asn                 45  #                 50 Met Lys Val Leu Tyr Asp Asp Asn His Val                55   #                 60Ser Ala Ile Asn Val Lys Ser Ile Asp Gln                 65  #                 70 Phe Arg Tyr Phe Asp Leu Ile Tyr Ser Ile                75   #                 80Lys Asp Thr Lys Leu Gly Asn Tyr Asp Asn                 85  #                 90 Val Arg Val Glu Phe Lys Asn Lys Asp Leu                95   #                100Ala Asp Lys Tyr Lys Asp Lys Tyr Val Asp                 105  #               110 Val Phe Gly Ala Asn Ala Tyr Tyr Gln Cys                115   #               120Ala Phe Ser Lys Lys Thr Asn Asp Ile Asn                 125  #               130 Ser His Gln Thr Asp Lys Arg Lys Thr Cys                135   #               140Met Tyr Gly Gly Val Thr Glu His Asn Gly                 145  #               150 Asn Gln Leu Asp Lys Tyr Arg Ser Ile Thr                155   #               160Val Arg Val Phe Glu Asp Gly Lys Asn Leu                 165  #               170 Leu Ser Phe Asp Val Gln Tyr Asn Lys Lys                175   #               180Lys Val Thr Ala Gln Glu Leu Asp Tyr Leu                 185  #               190 Thr Arg His Tyr Leu Val Lys Asn Lys Lys                195   #               200Leu Tyr Glu Phe Asn Asn Ser Pro Tyr Glu                 205  #               210 Thr Gly Tyr Ile Lys Phe Ile Glu Asn Glu                215   #               220Asn Ser Phe Trp Tyr Asp Met Met Pro Ala                 225  #               230 Pro Gly Asp Lys Phe Asp Gln Ser Lys Tyr                235   #               240Leu Met Met Tyr Asn Asp Asn Lys Met Val                 245  #               250 Asp Ser Lys Asp Val Lys Ile Glu Val Tyr                255   #               260 Leu Thr Thr Lys Lys Lys                265 (2) INFORMATION FOR SEQ ID NO:9:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1388          (B) TYPE: Nucleic Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:ATGAGTCAAC CAGATCCTAA ACCAGATGAG TTGCACAAAT      #                  #    40 CGAGTAAATT CACTGGTTTG ATGGAAAATA TGAAAGTTTT     #                   #    80GTATGATGAT AATCATGTAT CAGCAATAAA CGTTAAATCT      #                  #   120 ATAGATCAAT TTCGATACTT TGACTTAATA TATTCTATTA     #                   #   160AGGACACTAA GTTAGGGAAT TATGATAATG TTCGAGTCGA      #                  #   200 ATTTAAAAAC AAAGATTTAG CTGATAAATA CAAAGATAAA     #                   #   240TACGTAGATG TGTTTGGAGC TAATGCTTAT TATCAATGTG      #                  #   280 CTTTTTCTAA AAAAACGAAT GATATTAATT CGCATCAAAC     #                   #   320TGACAAACGA AAAACTTGTA TGTATGGTGG TGTAACTGAG      #                  #   360 CATAATGGAA ACCAATTAGA TAAATATAGA AGTATTACTG     #                   #   400TTCGGGTATT TGAAGATGGT AAAAATTTAT TATCTTTTGA      #                  #   440 CGTACAAACT AATAAGAAAA AGGTGACTGC TCAAGAATTA     #                   #   480GATTACCTAA CTCGTCACTA TTTGGTGAAA AATAAAAAAC      #                  #   520 TCTATGAATT TAACAACTCG CCTTATGAAA CGGGATATAT     #                   #   560TAAATTTATA GAAAATGAGA ATAGCTTTTG GTATGACATG      #                  #   600 ATGCCTGCAC CAGGAGATAA ATTTGACCAA TCTAAATATT     #                   #   640TAATGATGTA CAATGACAAT AAAATGGTTG ATTCTAAAGA      #                  #   680 TGTGAAGATT GAAGTTTATC TTACGACAAA GAAAAAGTGA     #                   #   720AATTATATTT TAGAAAAGTA AATATGAAGA GTTAGTAATT      #                  #   760 AAGGCAGGCA CTTATAGAGT ACCTGCCTTT TCTAATATTA     #                   #   800TTTAGTTATA GTTATTTTTG TTATATCTCT CTGATTTAGC      #                  #   840 ATTAACCCCT TGTTGCCATT ATAGTTTTCAC CAACTTTAG     #                   #   880CTGAAATTGG GGGATCATTT TTATCTTTAC TATGGATAGT      #                  #   920 TACTGTGTCG CCGTTTTTAA CGATTTGTTT CTCTTTTAAT     #                   #   960TTGTCAGTTA ATTTTTTCCA TGCATCATTT GCGTCAAACC      #                  #  1000 TATTTCCATT TGGATTTATT CTTGACAAAT CAATTCTTTT     #                   #  1040AACACTATCG GTATTAATCG GCTTGTTATT AAAATTACTA      #                  #  1080 AGTTCATCTA AATCAGCTGT ACCCGTAATA CTACTTTCGC     #                   #  1120CACCATTATT TAAATTGTAC GTAACACCAA CTGTCTCATT      #                  #  1160 TGCTGTTTTA TCGATAATAT TTGCTTCTTT CAAAGCATCT     #                   #  1200CTTACATTTT TCCATAAGTC TCTATCTGTT ATTTCAGAAG      #                  #  1240 CCTTTGCAAC GTTATTAATA CCATTATAAT TTGAAGAAGA     #                   #  1280ATGAAAACCT GAACCTACTG TTGTTAAAAC TAAAGCACTT      #                  #  1320 GCTATCAATG TTCTTGTTAA TAGTTTTTTA TTCATTTTAT     #                   #  1360 TTTCTCCTAT AACTTATTTG CAATCGAT         #                   #           1388 (2) INFORMATION FOR SEQ ID NO:10:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 239          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:Met Ser Gln Pro Asp Pro Lys Pro Asp Glu                  5 #                   #10 Leu His Lys Ser Ser Lys Phe Thr Gly Leu                15   #                 20Met Glu Asn Met Lys Val Leu Tyr Asp Asp                 25  #                 30 Asn His Val Ser Ala Ile Asn Val Lys Ser                35   #                 40Ile Asp Gln Phe Arg Tyr Phe Asp Leu Ile                 45  #                 50 Tyr Ser Ile Lys Asp Thr Lys Leu Gly Asn                55   #                 60Tyr Asp Asn Val Arg Val Glu Phe Lys Asn                 65  #                 70 Lys Asp Leu Ala Asp Lys Tyr Lys Asp Lys                75   #                 80Tyr Val Asp Val Phe Gly Ala Asn Ala Tyr                 85  #                 90 Tyr Gln Cys Ala Phe Ser Lys Lys Thr Asn                95   #                100Asp Ile Asn Ser His Gln Thr Asp Lys Arg                 105  #               110 Lys Thr Cys Met Tyr Gly Gly Val Thr Glu                115   #               120His Asn Gly Asn Gln Leu Asp Lys Tyr Arg                 125  #               130 Ser Ile Thr Val Arg Val Phe Glu Asp Gly                135   #               140Lys Asn Leu Leu Ser Phe Asp Val Gln Thr                 145  #               150 Asn Lys Lys Lys Val Thr Ala Gln Glu Leu                155   #               160Asp Tyr Leu Thr Arg His Tyr Leu Val Lys                 165  #               170 Asn Lys Lys Leu Tyr Glu Phe Asn Asn Ser                175   #               180Pro Tyr Glu Thr Gly Tyr Ile Lys Phe Ile                 185  #               190 Glu Asn Glu Asn Ser Phe Trp Tyr Asp Met                195   #               200Met Pro Ala Pro Gly Asp Lys Phe Asp Gln                 205  #               210 Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn                215   #               220Lys Met Val Asp Ser Lys Asp Val Lys Ile                 225  #               230 Glu Val Tyr Leu Thr Thr Lys Lys Lys                235 (2) INFORMATION FOR SEQ ID NO:11:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 731          (B) TYPE: Nucleic Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:TAAGGAGAAT TAAAAATGAA TAAAAAATTA CTAATGAATT      #                  #    40 TTTTTATCGT AAGCCCTTTG TTGCTTGCGA CAACTGCTAC     #                   #    80AGATTTTACC CCTGTTCCCT TATCATCTAA TCAAATAATC      #                  #   120 AAAACTGCAA AAGCATCTAC AAACGATAAT ATAAAGGATT     #                   #   160TGCTAGACTG GTATAGTAGT GGGTCTGACA CTTTTACAAA      #                  #   200 TAGTGAAGTT TTAGATAATT CCAGAGGATC TATGCGTATA     #                   #   240AAAAACACAG ATGGCAGCAT CAGCTTGATA ATTTTTCCGA      #                  #   280 GTCCTTATTA TAGCCCTGCT TTTACAAAAG GGGAAAAAGT     #                   #   320TGACTTAAAC ACAAAAAGAA CTAAAAAAAG CCAACATACT      #                  #   360 AGCGAAGGAA CTTATATCCA TTTCCAAATA AGTGGCGTTA     #                   #   400CAAATACTGA AAAATTACCT ACTCCAATAG AACTACCTTT      #                  #   440 AAAAGTTAAG GTTCATGGTA AAGATAGCCC CTTAAAGTAT     #                   #   480GGGCCAAAGT TCGATAAAAA ACAATTAGCT ATATCAACTT      #                  #   520 TAGACTTTGA AATTCGTCAT CAGCTAACTC AAATACATGG     #                   #   560ATTATATCGT TCAAGCGATA AAACGGGTGG TTATTGGAAA      #                  #   600 ATAACAATGA ATGACGGATC CACATATCAA AGTGATTTAT     #                   #   640CTAAAAAGTT TGAATACAAT ACTGAAAAAC CACCTATAAA      #                  #   680 TATTGATGAA ATAAAAACTA TAGAAGCAGA AATTAATTAA     #                   #   720 TTTACCACTT T               #                   #                   #      731(2) INFORMATION FOR SEQ ID NO:12:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 234           (B) TYPE: Amino Acid          (C) STRANDEDNESS: Unknown           (D) TOPOLOGY: Unknown    (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:Met Asn Lys Lys Leu Leu Met Asn Phe Phe                  5 #                   #10 Ile Val Ser Pro Leu Leu Leu Ala Thr Thr                15   #                 20Ala Thr Asp Phe Thr Pro Val Pro Leu Ser                 25  #                 30 Ser Asn Gln Ile Ile Lys Thr Ala Lys Ala                35   #                 40Ser Thr Asn Asp Asn Ile Lys Asp Leu Leu                 45  #                 50 Asp Trp Tyr Ser Ser Gly Ser Asp Thr Phe                55   #                 60Thr Asn Ser Glu Val Leu Asp Asn Ser Arg                 65  #                 70 Gly Ser Met Arg Ile Lys Asn Thr Asp Gly                75   #                 80Ser Ile Ser Leu Ile Ile Phe Pro Ser Pro                 85  #                 90 Tyr Tyr Ser Pro Ala Phe Thr Lys Gly Glu                95   #                100Lys Val Asp Leu Asn Thr Lys Arg Thr Lys                 105  #               110 Lys Ser Gln His Thr Ser Glu Gly Thr Tyr                115   #               120Ile His Phe Gln Ile Ser Gly Val Thr Asn                 125  #               130 Thr Glu Lys Leu Pro Thr Pro Ile Glu Leu                135   #               140Pro Leu Lys Val Lys Val His Gly Lys Asp                 145  #               150 Ser Pro Leu Lys Tyr Gly Pro Lys Phe Asp                155   #               160Lys Lys Gln Leu Ala Ile Ser Thr Leu Asp                 165  #               170 Phe Glu Ile Arg His Gln Leu Thr Gln Ile                175   #               180His Gly Leu Tyr Arg Ser Ser Asp Lys Thr                 185  #               190 Gly Gly Tyr Trp Lys Ile Thr Met Asn Asp                195   #               200Gly Ser Thr Tyr Gln Ser Asp Leu Ser Lys                 205  #               210 Lys Phe Glu Tyr Asn Thr Glu Lys Pro Pro                215   #               220Ile Asn Ile Asp Glu Ile Lys Thr Ile Glu                 225  #               230 Ala Glu Ile Asn (2) INFORMATION FOR SEQ ID NO:13:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1095          (B) TYPE: Nucleic Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:ATCATTAAAT ATAATTAATT TTCTTTTAAT ATTTTTTTAA      #                  #    40 TTGAATATTT AAGATTATAA GATATATTTA AAGTGTATCT     #                   #    80AGATACTTTT TGGGAATGTT GGATGAAGGA GATAAAAATG      #                  #   120 AATAAGAGTC GATTTATTTC ATGCGTAATT TTGATATTCG     #                   #   160CACTTATACT AGTTCTTTTT ACACCCAACG TATTAGCAGA      #                  #   200 GAGCCAACCA GACCCTACGC CAGATGAGTT GCACAAAGCG     #                   #   240AGTAAATTCA CTGGTTTGAT GGAAAATATG AAAGTTTTAT      #                  #   280 ATGATGATCA TTATGTATCA GCAACTAAAG TTAAGTCTGT     #                   #   320AGATAAATTT AGGGCACATG ATTTAATTTA TAACATTAGT      #                  #   360 GATAAAAAAC TGAAAAATTA TGACAAAGTG AAAACAGAGT     #                   #   400TATTAAATGA AGGTTTAGCA AAGAAGTACA AAGATGAAGT      #                  #   440 AGTTGATGTG TATGGATCAA ATTACTATGT AAACTGCTAT     #                   #   480TTTTCATCCA AAGATAATGT AGGTAAAGTT ACAGGTGGCA      #                  #   520 AAACTTGTAT GTATGGAGGA ATAACAAAAC ATGAAGGAAA     #                   #   560CCACTTTGAT AATGGGAACT TACAAAATGT ACTTATAAGA      #                  #   600 GTTTATGAAA ATAAAAGAAA CACAATTTCT TTTGAAGTGC     #                   #   640AAACTGATAA GAAAAGTGTA ACAGCTCAAG AACTAGACAT      #                  #   680 AAAAGCTAGG AATTTTTTAA TTAATAAAAA AAATTTGTAT     #                   #   720GAGTTTAACA GTTCACCATA TGAAACAGGA TATATAAAAT      #                  #   760 TTATTGAAAA TAACGGCAAT ACTTTTTGGT ATGATATGAT     #                   #   800GCCTGCACCA GGCGATAAGT TTGACCAATC TAAATATTTA      #                  #   840 ATGATGTACA ACGACAATAA AACGGTTGAT TCTAAAAGTG     #                   #   880TGAAGATAGA AGTCCACCTT ACAACAAAGA ATGGATAATG      #                  #   920 TTAATCCGAT TTTGATATAA AAAGTGAAAG TATTAGATAT     #                   #   960ATTTGAAAGG TAAGTACTTC GGTGCTTGCC TTTTTAGGAT      #                  #  1000 GCATATATAT AGATTAAACC GCACTTCTAT ATTAATAGAA     #                   #  1040AGTGCGGTTA TTTATACACT CAATCTAAAC TATAATAATT      #                  #  1080 GGAATCATCT TCAAA               #                  #                   #  1095 (2) INFORMATION FOR SEQ ID NO:14:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 266          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:Met Asn Lys Ser Arg Phe Ile Ser Cys Val                  5 #                   #10 Ile Leu Ile Phe Ala Leu Ile Leu Val Leu                15   #                 20Phe Thr Pro Asn Val Leu Ala Glu Ser Gln                 25  #                 30 Pro Asp Pro Thr Pro Asp Glu Leu His Lys                35   #                 40Ala Ser Lys Phe Thr Gly Leu Met Glu Asn                 45  #                 50 Met Lys Val Leu Tyr Asp Asp His Tyr Val                55   #                 60Ser Ala Thr Lys Val Lys Ser Val Asp Lys                 65  #                 70 Phe Arg Ala His Asp Leu Ile Tyr Asn Ile                75   #                 80Ser Asp Lys Lys Leu Lys Asn Tyr Asp Lys                 85  #                 90 Val Lys Thr Glu Leu Leu Asn Glu Gly Leu                95   #                100Ala Lys Lys Tyr Lys Asp Glu Val Val Asp                 105  #               110 Val Tyr Gly Ser Asn Tyr Tyr Val Asn Cys                115   #               120Tyr Phe Ser Ser Lys Asp Asn Val Gly Lys                 125  #               130 Val Thr Gly Gly Lys Thr Cys Met Tyr Gly                135   #               140Gly Ile Thr Lys His Glu Gly Asn His Phe                 145  #               150 Asp Asn Gly Asn Leu Gln Asn Val Leu Ile                155   #               160Arg Val Tyr Glu Asn Lys Arg Asn Thr Ile                 165  #               170 Ser Phe Glu Val Gln Thr Asp Lys Lys Ser                175   #               180Val Thr Ala Gln Glu Leu Asp Ile Lys Ala                 185  #               190 Arg Asn Phe Leu Ile Asn Lys Lys Asn Leu                195   #               200Tyr Glu Phe Asn Ser Ser Phe Tyr Glu Thr                 205  #               210 Gly Tyr Ile Lys Phe Ile Glu Asn Asn Gly                215   #               220Asn Thr Phe Trp Tyr Asp Met Met Pro Ala                 225  #               230 Pro Gly Asp Lys Phe Asp Gln Ser Lys Tyr                235   #               240Leu Met Met Tyr Asn Asp Asn Lys Thr Val                 245  #               250 Asp Ser Lys Ser Val Lys Ile Glu Val His                255   #               260 Leu Thr Thr Lys Asn Gly                265 (2) INFORMATION FOR SEQ ID NO:15:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1837          (B) TYPE: Nucleic Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:TCATGTTTGA CAGCTTATCA TCGATAAGCT TACTTTTCGA      #                  #    40 ATCAGGTCTA TCCTTGAAAC AGGTGCAACA TAGATTAGGG     #                   #    80CATGGAGATT TACCAGACAA CTATGAACGT ATATACTCAC      #                  #   120 ATCACGCAAT CGGCAATTGA TGACATTGGA ACTAAATTCA     #                   #   160ATCAATTTGT TACTAACAAG CAACTAGATT GACAACTAAT      #                  #   200 TCTCAACAAA CGTTAATTTA ACAACATTCA AGTAACTCCC     #                   #   240ACCAGCTCCA TCAATGCTTA CCGTAAGTAA TCATAACTTA      #                  #   280 CTAAAACCTT GTTACATCAA GGTTTTTTCT TTTTGTCTTG     #                   #   320TTCATGAGTT ACCATAACTT TCTATATTAT TGACAACTAA      #                  #   360 ATTGACAACT CTTCAATTAT TTTTCTGTCT ACTCAAAGTT     #                   #   400TTCTTCATTT GATATAGTCT AATTCCACCA TCACTTCTTC      #                  #   440 CACTCTCTCT ACCGTCACAA CTTCATCATC TCTCACTTTT     #                   #   480TCGTGTGGTA ACACATAATC AAATATCTTT CCGTTTTTAC      #                  #   520 GCACTATCGC TACTGTGTCA CCTAAAATAT ACCCCTTATC     #                   #   560AATCGCTTCT TTAAACTCAT CTATATATAA CATATTTCAT      #                  #   600 CCTCCTACCT ATCTATTCGT AAAAAGATAA AAATAACTAT     #                   #   640TGTTTTTTTT GTTATTTTAT AATAAAATTA TTAATATAAG      #                  #   680 TTAATGTTTT TTAAAAATAT ACAATTTTAT TCTATTTATA     #                   #   720GTTAGCTATT TTTTCATTGT TAGTAATATT GGTGAATTGT      #                  #   760 AATAACCTTT TTAAATCTAG AGGAGAACCC AGATATAAAA     #                   #   800TGGAGGAATA TTAATGGAAA ACAATAAAAA AGTATTGAAG      #                  #   840 AAAATGGTAT TTTTTGTTTT AGTGACATTT CTTGGACTAA     #                   #   880CAATCTCGCA AGAGGTATTT GCTCAACAAG ACCCCGATCC      #                  #   920 AAGCCAACTT CACAGATCTA GTTTAGTTAA AAACCTTCAA     #                   #   960AATATATATT TTCTTTATGA GGGTGACCCT GTTACTCACG      #                  #  1000 AGAATGTGAA ATCTGTTGAT CAACTTAGAT CTCACGATTT     #                   #  1040AATATATAAT GTTTCAGGGC CAAATTATGA TAAATTAAAA      #                  #  1080 ACTGAACTTA AGAACCAAGA GATGGCAACT TTATTTAAGG     #                   #  1120ATAAAAACGT TGATATTTAT GGTGTAGAAT ATTACCATCT      #                  #  1160 CTGTTATTTA TGTGAAAATG CAGAAAGGAG TGCATGTATC     #                   #  1200TACGGAGGGG TAACAAATCA TGAAGGGAAT CATTTAGAAA      #                  #  1240 TTCCTAAAAA GATAGTCGTT AAAGTATCAA TCGATGGTAT     #                   #  1280CCAAAGCCTA TCATTTGATA TTGAAACAAA TAAAAAAATG      #                  #  1320 GTAACTGCTC AAGAATTAGA CTATAAAGTT AGAAAATATC     #                   #  1360TTACAGATAA TAAGCAACTA TATACTAATG GACCTTCTAA      #                  #  1400 ATATGAAACT GGATATATAA AGTTCATACC TAAGAATAAA     #                   #  1440GAAAGTTTTT GGTTTGATTT TTTCCCTGAA CCAGAATTTA      #                  #  1480 CTCAATCTAA ATATCTTATG ATATATAAAG ATAATGAAAC     #                   #  1520GCTTGACTCA AACACAAGCC AAATTGAAGT CTACCTAACA      #                  #  1560 ACCAAGTAAC TTTTTGCTTT TGGCAACCTT ACCTACTGCT     #                   #  1600GGATTTAGAA ATTTTATTGC AATTCTTTTA TTAATGTAAA      #                  #  1640 AACCGCTCAT TTGATGAGCG GTTTTGTCTT ATCTAAAGGA     #                   #  1680GCTTTACCTC CTAATGCTGC AAAATTTTAA ATGTTGGATT      #                  #  1720 TTTGTATTTG TCTATTGTAT TTGATGGGTA ATCCCATTTT     #                   #  1760TCGACAGACA TCGTCGTGCC ACCTCTAACA CCAAAATCAT      #                  #  1800 AGACAGGAGC TTGTAGCTTA GCAACTATTT TATCGTC      #                   #    1837     (2) INFORMATION FOR SEQ ID NO:16:         (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 251    #          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:Met Glu Asn Asn Lys Lys Val Leu Lys Lys                  5 #                   #10 Met Val Phe Phe Val Leu Val Thr Phe Leu                15   #                 20Gly Leu Thr Ile Ser Gln Glu Val Phe Ala                 25  #                 30 Gln Gln Asp Pro Asp Pro Ser Gln Leu His                35   #                 40Arg Ser Ser Leu Val Lys Asn Leu Gln Asn                 45  #                 50 Ile Tyr Phe Leu Tyr Glu Gly Asp Pro Val                55   #                 60Thr His Glu Asn Val Lys Ser Val Asp Gln                 65  #                 70 Leu Arg Ser His Asp Leu Ile Tyr Asn Val                75   #                 80Ser Gly Pro Asn Tyr Asp Lys Leu Lys Thr                 85  #                 90 Glu Leu Lys Asn Gln Glu Met Ala Thr Leu                95   #                100Phe Lys Asp Lys Asn Val Asp Ile Tyr Gly                 105  #               110 Val Glu Tyr Tyr His Leu Cys Tyr Leu Cys                115   #               120Glu Asn Ala Glu Arg Ser Ala Cys Ile Tyr                 125  #               130 Gly Gly Val Thr Asn His Glu Gly Asn His                135   #               140Leu Glu Ile Pro Lys Lys Ile Val Val Lys                 145  #               150 Val Ser Ile Asp Gly Ile Gln Ser Leu Ser                155   #               160Phe Asp Ile Glu Thr Asn Lys Lys Met Val                 165  #               170 Thr Ala Gln Glu Leu Asp Tyr Lys Val Arg                175   #               180Lys Tyr Leu Thr Asp Asn Lys Gln Leu Tyr                 185  #               190 Thr Asn Gly Pro Ser Lys Tyr Glu Thr Gly                195   #               200Tyr Ile Lys Phe Ile Pro Lys Asn Lys Glu                 205  #               210 Ser Phe Trp Phe Asp Phe Phe Pro Glu Pro                215   #               220Glu Phe Thr Gln Ser Lys Tyr Leu Met Ile                 225  #               230 Tyr Lys Asp Asn Glu Thr Leu Asp Ser Asn                235   #               240Thr Ser Gln Ile Glu Val Tyr Leu Thr Thr                 245  #               250 Lys (2) INFORMATION FOR SEQ ID NO:17:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 82          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:17:Ser His Asp Gln Phe Leu Gln His Thr Ile 5                   # 10Leu Phe Lys Gly Phe Phe Thr Asp His Ser 15                   # 20Trp Tyr Asn Asp Leu Leu Val Asp Phe Asp 25                   # 30Ser Lys Asp Ile Val Asp Lys Tyr Lys Gly 35                   # 40Lys Lys Val Asp Leu Tyr Gly Ala Tyr Tyr 45                   # 50Gly Tyr Gln Cys Ala Gly Gly Thr Pro Asn 55                   # 60Lys Thr Ala Cys Met Tyr Gly Gly Val Thr 65                   # 70Leu His Asp Asn Asn Arg Leu Thr Glu Glu 75                   # 80Lys Lys (2) INFORMATION FOR SEQ ID NO:18:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 82          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:18:Thr Gly Asp Gln Phe Leu Glu Asn Thr Leu 5                   # 10Leu Tyr Lys Lys Phe Phe Thr Asp Leu Ile 15                   # 20Asn Phe Glu Asp Leu Leu Ile Asn Phe Asn 25                   # 30Ser Lys Glu Met Ala Gln His Phe Lys Ser 35                   # 40Lys Asn Val Asp Val Tyr Pro Ile Arg Tyr 45                   # 50Ser Ile Asn Cys Tyr Gly Gly Glu Ile Asp 55                   # 60Arg Thr Ala Cys Thr Tyr Gly Gly Val Thr 65                   # 70Pro His Glu Gly Asn Lys Leu Lys Glu Arg 75                   # 80Lys Lys (2) INFORMATION FOR SEQ ID NO:19:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 82          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:19:Ser Asp Asp Gln Phe Leu Glu Asn Thr Leu 5                   # 10Leu Phe Lys Gly Phe Phe Thr Gly His Pro 15                   # 20Trp Tyr Asn Asp Leu Leu Val Asp Leu Gly 25                   # 30Ser Lys Asp Ala Thr Asn Lys Tyr Lys Gly 35                   # 40Lys Lys Val Asp Leu Tyr Gly Ala Tyr Tyr 45                   # 50Gly Tyr Gln Cys Ala Gly Gly Thr Pro Asn 55                   # 60Lys Thr Ala Cys Met Tyr Gly Gly Val Thr 65                   # 70Leu His Asp Asn Asn Arg Leu Thr Glu Glu 75                   # 80Lys Lys (2) INFORMATION FOR SEQ ID NO:20:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 89          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:20:Ser Ile Asp Gln Phe Leu Tyr Phe Asp Leu 5                   # 10Ile Tyr Ser Ile Lys Asp Thr Lys Leu Gly 15                   # 20Asn Tyr Asp Asn Val Arg Val Glu Phe Lys 25                   # 30Asn Lys Asp Leu Ala Asp Lys Tyr Lys Asp 35                   # 40Lys Tyr Val Asp Val Phe Gly Ala Asn Tyr 45                   # 50Tyr Gln Cys Tyr Phe Ser Lys Lys Thr Asn 55                   # 60Asp Ile Asn Ser His Gln Thr Asp Lys Arg 65                   # 70Lys Thr Cys Met Tyr Gly Gly Val Thr Glu 75                   # 80His Asn Gly Asn Gln Leu Asp Lys Tyr 85 (2) INFORMATION FOR SEQ ID NO:21:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 89          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:21:Ser Val Asp Lys Phe Leu Ala His Asp Leu 5                   # 10Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys 15                   # 20Asn Tyr Asp Lys Val Lys Thr Glu Leu Leu 25                   # 30Asn Glu Gly Leu Ala Lys Lys Tyr Lys Asp 35                   # 40Glu Val Val Asp Val Tyr Gly Ser Asn Tyr 45                   # 50Tyr Val Asn Cys Tyr Phe Ser Ser Lys Asp 55                   # 60Asn Val Gly Lys Val Thr Gly Gly Lys Thr 65                   # 70Cys Met Tyr Gly Gly Ile Thr Lys His Glu 75                   # 80Gly Asn His Phe Asp Asn Gly Asn Leu 85 (2) INFORMATION FOR SEQ ID NO:22:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 89          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:22:Ser Val Asp Lys Phe Leu Ala His Asp Leu 5                   # 10Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys 15                   # 20Asn Tyr Asp Lys Val Lys Thr Glu Leu Leu 25                   # 30Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp 35                   # 40Glu Val Val Asp Val Tyr Gly Ser Asn Tyr 45                   # 50Tyr Val Asn Cys Tyr Phe Ser Ser Lys Asp 55                   # 60Asn Val Gly Lys Val Thr Gly Gly Lys Thr 65                   # 70Cys Met Tyr Gly Gly Ile Thr Lys His Glu 75                   # 80Gly Asn His Phe Asp Asn Gly Asn Leu 85 (2) INFORMATION FOR SEQ ID NO:23:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH:89          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:23:Ser Val Asp Lys Phe Leu Ala His Asp Leu 5                   # 10Ile Tyr Asn Ile Ser Asp Lys Lys Leu Lys 15                   # 20Asn Tyr Asp Lys Val Lys Thr Glu Leu Leu 25                   # 30Asn Glu Asp Leu Ala Lys Lys Tyr Lys Asp 35                   # 40Glu Val Val Asp Val Tyr Gly Ser Asn Tyr 45                   # 50Tyr Val Asn Cys Tyr Phe Ser Ser Lys Asp 55                   # 60Asn Val Gly Lys Val Thr Gly Gly Lys Thr 65                   # 70Cys Met Tyr Gly Gly Ile Thr Lys His Glu 75                   # 80Gly Asn His Phe Asp Asn Gly Asn Leu 85 (2) INFORMATION FOR SEQ ID NO:24:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 79          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:24:Ser Val Asp Gln Leu Leu Ser His Asp Leu 5                   # 10Ile Tyr Asn Val Ser Gly Pro Asn Tyr Asp 15                   # 20Lys Leu Lys Thr Glu Leu Lys Asn Gln Glu 25                   # 30Met Ala Thr Leu Phe Lys Asp Lys Asn Val 35                   # 40Asp Ile Tyr Gly Val Glu Tyr Tyr His Leu 45                   # 50Cys Tyr Leu Cys Glu Asn Ala Glu Arg Ser 55                   # 60Ala Cys Ile Tyr Gly Gly Val Thr Asn His 65                   # 70Glu Gly Asn His Leu Glu Ile Pro Lys 75 (2) INFORMATION FOR SEQ ID NO:25:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 73          (B) TYPE: Amino Acid           (C) STRANDEDNESS: Unknown          (D) TOPOLOGY: Unknown     (ii) MOLECULE TYPE: Peptide    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:25:Val Leu Asp Asn Ser Leu Gly Ser Met Arg 5                   # 10Ile Lys Asn Thr Asp Gly Ser Ile Ser Leu 15                   # 20Ile Ile Phe Pro Ser Pro Tyr Tyr Ser Pro 25                   # 30Ala Phe Thr Lys Gly Glu Lys Val Asp Leu 35                   # 40Asn Thr Lys Arg Thr Lys Lys Ser Gln His 45                   # 50Thr Ser Glu Gly Thr Tyr Ile His Phe Gln 55                   # 60Ile Ser Gly Val Thr Asn Thr Glu Lys Leu 65                   # 70Pro Thr Pro

What is claimed is:
 1. An isolated and purified superantigen toxin DNAfragment encoding Staphylococcal enterotoxin B (SEB) in which at leastone amino acid selected from the group consisting of amino acidpositions 40-50, 62-72, 84-94 and 110-120 of SEB, and at least one aminoacid of amino acid positions 18-28, 55-65, 86-96, 89-99, and 205-215 ofSEB have been altered such that binding of said encoded SEB to the MHCclass II receptor and T cell antigen receptor is altered.
 2. An isolatedand purified superantigen toxin DNA fragment encoding Staphylococcalenterotoxin B (SEB), wherein said superantigen toxin is Staphylococcalenterotoxin B having the sequence of SEQ ID NO:5.
 3. An isolated andpurified superantigen toxin DNA fragment encoding Staphylococcalenterotoxin B (SEB), wherein said superantigen toxin is Staphylococcalenterotoxin B having the sequence of SEQ ID NO:7.
 4. An isolated andpurified DNA fragment superantigen toxin DNA fragment encodingStaphylococcal enterotoxin B (SEB), wherein said superantigen toxin isStaphylococcal enterotoxin B having the sequence of SEQ ID NO:9.
 5. Anisolated and purified DNA fragment superantigen toxin DNA fragmentencoding Staphylococcal enterotoxin B (SEB), wherein said fragmentencodes the amino acid sequence of SEQ ID NO:6.
 6. An isolated andpurified DNA fragment superantigen toxin DNA fragment encodingStaphylococcal enterotoxin B (SEB), wherein said fragment encodes theamino acid sequence of SEQ ID NO:8.
 7. An isolated and purified DNAfragment superantigen toxin DNA fragment encoding Staphylococcalenterotoxin B (SEB), wherein said fragment encodes the amino acidsequence of SEQ ID NO:10.
 8. A recombinant DNA construct comprising avector containing an isolated and purified altered Staphylococcalenterotoxin B superantigen toxin DNA fragment according to claim
 1. 9. Arecombinant DNA construct comprising a vector containing an isolated andpurified superantigen toxin DNA fragment encoding Staphylococcalenterotoxin B (SEB), wherein said DNA fragment has the sequenceaccording to sequence of SEQ ID NO:5.
 10. A recombinant DNA constructaccording to claim 9 wherein said construct is pETB2360210.
 11. Arecombinant DNA construct comprising a vector containing an isolated andpurified superantigen toxin DNA fragment encoding Staphylococcalenterotoxin B (SEB), wherein said DNA fragment has the sequenceaccording to sequence of SEQ ID NO:7.
 12. A recombinant DNA constructaccording to claim 11 wherein said construct pETB899445P.
 13. Arecombinant DNA construct comprising a vector containing an isolated andpurified superantigen toxin DNA fragment encoding Staphylococcalenterotoxin B (SEB), wherein said DNA fragment has the sequenceaccording to sequence of SEQ ID NO:9.
 14. A recombinant DNA constructaccording to claim 13 wherein said construct is pETB899445C.
 15. Therecombinant DNA construct according to claim 8, wherein said DNAfragment encodes the amino acids sequence specified in SEQ ID NO:6. 16.An isolated host cell transformed with a recombinant DNA constructaccording to claim
 15. 17. A host cell according to claim 16, whereinsaid cell is prokaryotic.
 18. A method for producing alteredsuperantigen toxin comprising culturing the cells according to claim 16,under conditions such that said DNA fragment is expressed and saidsuperantigen toxin is thereby produced, and isolating said superantigentoxin.
 19. The recombinant DNA construct according to claim 8, whereinsaid DNA fragment encodes the amino acids sequence specified in SEQ IDNO:8.
 20. An isolated host cell transformed with a recombinant DNAconstruct according to claim
 19. 21. A host cell according to claim 20,wherein said cell is prokaryotic.
 22. A method for producing alteredsuperantigen toxin comprising culturing the cells according to claim 20,under conditions such that said DNA fragment is expressed and saidsuperantigen toxin is thereby produced, and isolating said superantigentoxin.
 23. The recombinant DNA construct according to claim 8, whereinsaid DNA fragment encodes the amino acids sequence specified in SEQ IDNO:10.
 24. An isolated host cell transformed with a recombinant DNAconstruct according to claim
 23. 25. A host cell according to claim 24,wherein said cell is prokaryotic.
 26. A method for producing alteredsuperantigen toxin comprising culturing the cells according to claim 24,under conditions such that said DNA fragment is expressed and saidsuperantigen toxin is thereby produced, and isolating said superantigentoxin.
 27. A recombinant DNA construct according to claim 8, whereinsaid vector is an expression vector.
 28. An isolated host celltransformed with a recombinant DNA construct according to claim
 8. 29. Ahost cell according to claim 28, wherein said cell is prokaryotic.
 30. Amethod for producing altered superantigen toxin comprising culturing thecells according to claim 28, under conditions such that said DNAfragment is expressed and said superantigen toxin is thereby produced,and isolating said superantigen toxin.